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Chronic endothelin-1-induced changes in vascular reactivity in rat resistance arteries and aorta
European Journal of Pharmacology 359 Ž1998. 69–75
Chronic endothelin-1-induced changes in vascular reactivity in rat resistance arteries and aorta Marc Iglarz, Bernard I. Levy, ´ Daniel Henrion
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(INSERM) U 141, IFR Circulation-Lariboisiere, Institut National de la Sante´ et de la Recherche Medicale Lariboisiere, ´ ` UniÕersite´ Paris VII, Hopital ˆ ` 41 bd de la Chapelle 75475 Paris, France Received 27 March 1998; revised 17 August 1998; accepted 18 August 1998
Abstract The role of endothelin-1 in vascular homeostasis is not yet clearly established. We investigated the responses to phenylephrine and acetylcholine in rat mesenteric resistance artery and aorta mounted in vitro in myographs after a 2-week treatment with endothelin-1 Ž5 pmol kgy1 miny1 , n s 8.. Systolic arterial blood pressure increased in endothelin-1-treated rats Ž171 " 7 mmHg vs. 196 " 6 mmHg, P - 0.05.. In the aorta, chronic endothelin-1 significantly increased the dilator response to acetylcholine Žmaximal dilatation: 76 " 3 vs. 86 " 3% in control, P - 0.05.. Acetylcholine-induced dilatation was decreased by nitric oxide ŽNO. synthase inhibition with N G-nitroL-arginine methyl ester ŽL-NAME 100 mmolrl. and partly restored by cyclooxygenases inhibition Žindomethacin, 10 mmolrl.. In endothelin-1-treated rats, L-NAME-sensitive acetylcholine dilatation was lower than in the control, but dilator cyclooxygenase productŽs. were found instead of constrictor cyclooxygenase productŽs.. In mesenteric resistance arteries chronic endothelin-1 increased the participation of cyclooxygenase products in acetylcholine-induced dilatation from 10 " 2 to 19 " 3%. In both types of arteries, phenylephrine-induced contraction was not affected by chronic endothelin-1. Thus chronic endothelin-1 increased the participation of dilator cyclooxygenase productŽs. in acetylcholine-induced dilatation in the aorta and the mesenteric resistance arteries. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Acetylcholine; Phenylephrine; Resistance artery; Aorta; Vascular reactivity; Indomethacin; N G -nitro-L-arginine methyl ester ŽL-NAME.; Endothelin; Hypertension
1. Introduction Endothelin-1 is a potent vasoconstrictor and mitogen produced by endothelial and vascular smooth muscle cells ŽYanagisawa et al., 1988.. This hormone exerts its biological effects via activation of specific endothelin-1 type A ŽETA . and type B ŽETB . receptors. Endothelin-1 type A receptors, which are expressed by vascular smooth muscle cells cause vasoconstriction ŽHaynes et al., 1995.. Endothelin-1 type B receptors expressed by vascular smooth muscle cells are also responsible for vasoconstriction and those expressed by endothelial cells can cause vasorelaxation ŽNakashima and Vanhoutte, 1993; Shetty et al., 1993.. The effects of endothelin-1 on vascular tone are different depending on species and vessel type. This reflects a difference in receptors distribution and a difference in the type of effectors produced upon endothelin-1 stimu) Corresponding author. Tel.: q33-1-4285-8672; Fax: q33-1-44631864; E-mail: [email protected]
lation. Endothelin-1-induced contraction may be mediated by cyclooxygenase ŽBarnett et al., 1994. or cytochrome P450 products ŽOyekan et al., 1997.. Endothelin-1-dependent vasorelaxation may also be mediated by one or several agents such as cyclooxygenase productŽs. ŽLal et al., 1996, Matsuda et al., 1993., nitric oxide ŽNO. ŽMagazine and Srivastava, 1996. and endothelium-derived hyperpolarizing factorŽs. ŽEDHF. ŽSakuma et al., 1993.. Endothelin-1 might be involved in several pathological situations such as heart failure ŽSakai et al., 1996., preeclampsia ŽGreer et al., 1991., renal dysfunction ŽRabelink et al., 1996; Karam et al., 1996., sepsis ŽSharma et al., 1997., atherosclerosis ŽHasdai et al., 1997., cerebral vasospasm ŽCosentino and Katusic, 1994; Zuccarello et al., 1996., in effects of aging ŽBattistelli et al., 1996. and in hypertension ŽSuzuki et al., 1990; Kohno et al., 1991; Lariviere et al., 1993; Li et al., 1996. or effects of chronic administration of angiotensin II ŽD’Uscio et al., 1997.. No study, however, has yet defined the changes in vascular reactivity due to chronic elevation of endothelin-1 in either
0014-2999r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 1 4 - 2 9 9 9 Ž 9 8 . 0 0 6 1 6 - 5
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large or in resistance vessels. Chronic infusion of endothelin-1 increases blood pressure ŽMortensen et al., 1990. and physiological concentrations of endothelin-1 raise the agonist-induced tone ŽHenrion and Laher, 1993.. Our working hypothesis was that chronic administration of endothelin-1 should change the vascular reactivity to both vasodilator and vasoconstrictor agents as endothelin-1 is involved in both dilator and constrictor processes Žsee above.. We investigated the impact of chronic endothelin-1 treatment on the vascular reactivity of large Žaorta. and small resistance arteries Žmesenteric. to phenylephrine and acetylcholine.
2. Materials and methods 2.1. Animal preparation Fifteen male Wistar–Kyoto rats ŽIffa-Credo, Lyon, France., 10-weeks-old and weighing 250 g, were randomly allocated to a control group Žfed with standard chow and tap water, n s 8. or to a group receiving endothelin-1 subcutaneously Ž5 pmol kgy1 miny1, osmotic minipumps Alzet, model 2002, Palo Alto, CA, USA, n s 7. for 2 weeks. After 2 weeks, systolic blood pressure was monitored by the tail cuff method ŽBP recorder 8006, Ugo Basile, Comerio, Italy.. The thoracic aorta and a section of the mesenteric bed were dissected out and immersed in ice-cold physiological salt solution Žcomposition described below.. The procedure followed for the care and killing of the study animals was in accordance with the European Community standards on the care and use of laboratory animals ŽMinistere ` de l’Agriculture, France, authorisation no. 00577.. 2.2. Isolated ring segments of aorta Ring segments of aorta, cleaned of fat and connective tissues, 3 mm in length, were mounted between two stainless-steel wires in 3-ml organ baths containing physiological salt solution of the following composition Žin mmolrl.: 135.0 NaCl, 15.0 NaHCO 3 , 4.6 KCl, 1.5 CaCl 2 , 1.2 MgSO4 , and 11.0 glucose. The solution was bubbled with 95% O 2 and 5% O 2 and the pH was 7.4. Physiological salt solutions containing Kq 15 or 60 mmolrl were prepared by equimolar substitution of KCl for NaCl. One wire was attached to a fixed support while the second wire was connected to a moveable holder supporting a tension transducer ŽGrass FT.03, Quincy, MA, USA. so that isometric force measurements could be collected by a Biopac data acquisition system ŽBiopac MP 100, LaJolla, CA, USA. and recorded by a computer ŽApple computers, Cupertino, CA, USA., using the Acknowledge w data acquisition and analysis software ŽBiopac, LaJolla, CA, USA.. The arterial segments were allowed to recover for 30 min. during which time the physiological salt solution
was replaced at 15-min intervals. Following this recovery period, stepwise increases in tension were applied to each segment and the response to KCl 60 mmolrl was determined at each level of tension. The optimal tension was determined as the tension corresponding to the optimal response to KCl. The average tension needed was 2 g, irrespective of the treatment Ž2.04 " 0.07 g, n s 32 segments in control and 1.93 " 0.09 g, n s 28 segments in endothelin-1-treated rats.. Segments of aorta were allowed to equilibrate for an additional 90 min. Four segments of aorta were isolated per rat and a concentration-response curve for one of the drugs described below was obtained. At the end of the experimental protocol aorta ring segments were blotted dry and weighed. Concentration–response curves for phenylephrine Ž1 nmolrl to 100 mmolrl. were obtained by cumulative addition of phenylephrine to the bath solution. The data are N force per g tissue. Concentration–response curves for acetylcholine Ž1 nmolrl to 10 mmolrl. were obtained by cumulative addition of acetylcholine to the physiological salt solution after preconstriction of the arterial segments with a concentration of phenylephrine Ž30 to 300 nmolrl. sufficient to reach approximately 70% of the tissue maximum as determined with Kq Ž60 mmolrl.. Data are expressed as % dilatation of phenylephrine-induced preconstriction. Concentration–response curves for phenylephrine and acetylcholine were repeated after incubation of the segments of aorta for 30 min. with the NO synthase inhibitor, N G -nitro-L-arginine methyl ester ŽL-NAME. 100 mmolrl, or L-NAME 100 mmolrlq the non-selective cyclooxygenase inhibitor indomethacin 10 mmolrl, or L-NAME 100 mmolrl q indomethacin 10 mmolrl q KCl 15 mmolrl. As NO synthesis blockade with L-NAME may not be complete ŽGriffith and Stuehr, 1995. we used a concentration 10 times higher than that in previous studies in which L-NAME has been shown to block NO-dependent responses ŽHenrion et al., 1997a,b. and one with an effect equivalent to that of another blocker ŽDowell et al., 1996.. No drug was added in the fourth segment of aorta before repetition of the concentration–response curve for phenylephrine and acetylcholine. This segment of aorta served as a time control. In another series of experiments, indomethacin 10 mmolrl or KCl 15 mmolrl was added to the bath solution in the absence of other substances. 2.3. Isolated mesenteric resistance arteries Segments of second-order mesenteric artery Žapprox. 200 mm external diameter. were trimmed free of fat and adhering connective tissue, and mounted in a myograph according to the technique of Mulvany and Halpern Ž1977.. Isometric tension was recorded and collected with a Biopac data acquisition system ŽBiopac MP 100. and recorded by a computer ŽApple computers. using the Acknowledge w
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reach approximately 70% of the tissue maximum as determined with Kq Ž60 mmolrl.. The data are expressed as % dilatation of phenylephrine-induced preconstriction. The tissues were allowed to equilibrate for 15 min between responses. Concentration–response curves to phenylephrine and acetylcholine were repeated after successive incubation of the segments of mesenteric arteries for 20 min with LNAME Ž100 mmolrl., indomethacin Ž10 mmolrl., and then KCl 15 mmolrl. In a separate series of experiments, 4 successive concentration–response curves to phenylephrine and acetylcholine were made with segments of mesenteric artery without the successive addition of LNAME Ž100 mmolrl., indomethacin Ž10 mmolrl., and KCl 15 mmolrl. This group of experiments served as a time control. In separate series of experiments indo-
Fig. 1. Concentration-dependent contraction in response to phenylephrine in rat aorta Župper panel. and mesenteric resistance artery segments Žlower panel.. Rats were treated for 2 weeks with endothelin-1 Ž5 pmol kgy1 miny1 , open circles. or with the solvent Žopen squares.. Data are given as the means"S.E.M. Two-factor ANOVA for consecutive measurements, endothelin-1 vs. control: no significant difference.
data acquisition and analysis software ŽBiopac.. Segments were allowed to equilibrate for 30 min in physiological salt solution, maintained at 378C and gassed with 95% O 2 and 5% CO 2 . The physiological salt solution was replaced every 15 min. Each vessel was placed under optimal stretch, equivalent to an in vivo arterial blood pressure of approximately 100 mmHg ŽMulvany and Halpern, 1977; Lew and McPherson, 1996.. The vessels were allowed to equilibrate for 30 min. Tissue contractility was then assessed by exposure to Kq Ž60 mmolrl.. Subsequently, cumulative concentration–response curves were obtained for phenylephrine Ž10 nmolrl to 100 mmolrl.. The data are expressed as mN force per mm vessel length. Acetylcholine concentration–response curves Ž1 nmolrl to 10 mmolrl. were obtained after a preconstriction of the mesenteric rings with a concentration of phenylephrine Ž10 to 100 nmolrl. sufficient to
Fig. 2. Concentration-dependent dilatation in response to acetylcholine in rat aorta Župper panel. and mesenteric resistance artery segments Žlower panel.. Rats were treated for 2 weeks with endothelin-1 Ž5 pmol kgy1 miny1 , open circles. or with the solvent Žopen squares.. Data are given as the means"S.E.M. aP - 0.05, two-factor ANOVA, endothelin-1 vs. control. ) P - 0.05, one-way ANOVA, endothelin-1 vs. control.
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where E is the contraction, Emax is the maximum contraction, C is the concentration, EC is the EC 50 ŽIC 50 for relaxation. Žconcentration of agonist required to induce half the maximum response. and m is the Hill coefficient. Comparisons between groups were made using a one factor analysis of variance ŽANOVA. followed by Dunnett’s t-test or by two-factor ANOVA for repeated measures to compare the concentration–response curves Žtreated animals versus controls or relaxation pathway inhibitors versus control.. A probability level of P - 0.05 was considered significant.
Fig. 3. Effect of the consecutive addition of L-NAME Ž100 mmolrl., indomethacin Ž10 mmolrl. and KCl Ž15 mmolrl. on the concentrationdependent dilatation in response to acetylcholine in aorta ring segments isolated from rats treated for 2 weeks with endothelin-1 Ž5 pmol kgy1 miny1 , lower panel. or with the solvent Župper panel.. Data are given as the means"S.E.M. Groups were control Žopen squares., L-NAME Žclosed squares., indomethacin Žclosed circles. and KCl Žopen circles.. ) P 0.05, one-way ANOVA, L-NAME versus control. §P - 0.05, one-way ANOVA, L-NAMEqindomethacin vs. L-NAME. aP - 0.05, one-way ANOVA, L-NAMEqindomethacinqKCl vs. L-NAMEqindomethacin. Two-factor ANOVA: L-NAME vs. control Ž P - 0.05, lower and upper panel., L-NAMEqindomethacin vs. control or L-NAME Ž P - 0.05, lower and upper panel. and L-NAMEqindomethacinqKCl vs. L-NAMEq indomethacin Ž P - 0.05, upper panel only..
methacin 10 mmolrl or KCl 15 mmolrl was added to the bath solution in the absence of other substances. 2.4. Statistical analysis The results are expressed as means " S.E.M. EC 50 ŽIC 50 for relaxation. and Emax were calculated individually for each concentration–response curve using the equation ŽMichaelis and Menten, 1913.: E s Ž Emax = C m . r Ž EC m q C m .
Fig. 4. Effect of the addition of L-NAME Ž100 mmolrl., indomethacin Ž10 mmolrl. and KCl Ž15 mmolrl. on the concentration-dependent dilatation in response to acetylcholine in mesenteric artery segments isolated from rats treated for 2 weeks with endothelin-1 Ž5 pmol kgy1 miny1 , lower panel. or with the solvent Župper panel.. Groups were control Žopen squares., L-NAME Žclosed squares., indomethacin Žclosed circles. and KCl Žopen circles.. Data are given as the means"S.E.M. ) P - 0.05, one-way ANOVA, L-NAME vs. control. §P - 0.05, one-way ANOVA, L-NAMEqindomethacin vs. L-NAME. aP - 0.05, one-way ANOVA, L-NAMEqindomethacinqKCl vs. L-NAMEqindomethacin. Two-factor ANOVA Ž P - 0.05, lower and upper panel.: L-NAME vs. control, L-NAMEqindomethacin versus control or L-NAME and LNAMEqindomethacinqKCl vs. L-NAMEqindomethacin.
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2.5. Drugs
3.3. Acetylcholine-induced dilatation
Endothelin-1, acetylcholine, phenylephrine, L-NAME and indomethacin were purchased from Sigma ŽSt. Louis, MO, USA.. The other reagents were obtained from Prolabo ŽParis, France..
Acetylcholine Ž1 nmolrl to 10 mmolrl. induced a concentration-dependent dilatation of aortic and mesenteric artery ring segments precontracted with phenylephrine ŽFig. 2.. Precontraction with phenylephrine was equivalent in all groups. Chronic endothelin-1 significantly increased acetylcholine-induced dilatation in the aorta ŽFig. 2; Emax s 76 " 3 vs. 86 " 3% relaxation, P - 0.05; no significant change in EC 50 : 29 " 7 nmolrl vs. 48 " 9 nmolrl.. The greatest increase in acetylcholine-induced dilatation was observed when acetylcholine was 30 nmolrl Ž42% increase in dilatation, Fig. 2.. On the other hand, chronic endothelin-1 did not significantly affect acetylcholine-induced dilatation in mesenteric resistance arteries ŽFig. 2.. The addition of L-NAME Ž100 mmolrl., indomethacin Ž10 mmolrl. or KCl Ž15 mmolrl. significantly attenuated acetylcholine-induced dilatation in aorta ŽFig. 3. and in mesenteric artery ring segments ŽFig. 4.. In the aorta, L-NAME Ž100 mmolrl. induced a significant attenuation of acetylcholine-induced dilatation ŽFig. 3.. Chronic endothelin-1 induced a significant change in L-NAME Ž100 mmolrl.-induced attenuation of acetylcholine dilatation ŽFigs. 3 and 5.. In the aorta, indomethacin Ž10 mmolrl., in addition to L-NAME, significantly increased acetylcholine-induced dilatation, reflecting the participation of vasoconstrictor cyclooxygenase productŽs. in the response to acetylcholine Žrepresenting 24 " 5% of the total response to acetylcholine, Fig. 5.. In contrast, in the aorta of rats treated chronically with endothelin-1, indomethacin Ž10 mmolrl., in addition to LNAME, further decreased acetylcholine-induced dilatation,
3. Results 3.1. Blood pressure A 2-week treatment with endothelin-1 significantly increased systolic arterial pressure Ž196 " 6 mm Hg vs. 171 " 7 in the control group, P - 0.05.. 3.2. Phenylephrine-induced contraction Phenylephrine induced a concentration-dependent contraction in aorta and mesenteric resistance arterial segments ŽFig. 1.. Chronic endothelin-1 had no significant effect on phenylephrine-induced contraction in either aorta or mesenteric arterial segments ŽFig. 1.. The consecutive addition of L-NAME Ž100 mM. and indomethacin Ž10 mM. did not significantly affect the response to phenylephrine in the aorta or in the mesenteric artery Ždata not shown.. KCl Ž15 mM. significantly increased Ž P - 0.005. the response to phenylephrine ŽEC 50 from 43 " 6 to 7.2 " 1.1 nmolrl in the aorta and from 1.1 " 0.16 to 0.18 " 0.05 mmolrl in the mesenteric artery; no effect of chronic endothelin-1..
Fig. 5. Effect of chronic endothelin-1 treatment on the distribution of vasoactive agents in the dilator response to acetylcholine in the aorta and in the mesenteric artery. Bars represent the mean percentage Ž"S.E.M.. of each agent calculated by measuring the areas under the curve ŽFig. 3Fig. 4. of each concentration–response curve to acetylcholine obtained after the successive addition of the inhibitors: L-NAME, indomethacin and then KCl. Data are given as the means and S.E.M. Žnot presented. are less than 10% of means. ) P - 0.05, one-way ANOVA, chronic endothelin-1 vs. control.
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reflecting the participation of vasodilator cyclooxygenase productŽs. in acetylcholine-induced dilatation Žrepresenting 40 " 6% of the total response to acetylcholine, Figs. 3 and 5.. The further addition of KCl Ž15 mmolrl., after L-NAME and indomethacin, abolished the residual acetylcholine-induced dilatation of aortic rings ŽFig. 3.. The effect of KCl Ž15 mmolrl. was significantly decreased after chronic endothelin-1 ŽFig. 5.. The effect of indomethacin or KCl Ž15 mmolrl. was identical to that described above when indomethacin or KCl Ž15 mmolrl. was used alone Ždata not shown.. In mesenteric resistance artery segments, the successive addition of L-NAME Ž100 mmolrl., indomethacin Ž10 mmolrl. and KCl Ž15 mmolrl. significantly attenuated acetylcholine-induced dilatation and allowed the differentiation of 3 distinct dilator pathways: NO, suppressed by L-NAME, cyclooxygenase productŽs., suppressed by indomethacin and EDHF, suppressed by KCl ŽFigs. 4 and 5.. Chronic endothelin-1 increased the participation of cyclooxygenase products in acetylcholine-induced dilatation from 10 " 2 to 19 " 3% ŽFig. 5.. In a separate group of experiments 4 successive concentration-response curves for acetylcholine were obtained with mesenteric resistance arteries, without the addition of L-NAME, indomethacin or KCl, in order to test the effect of time on the responses. The 4 successive curves had similar Emax Ž98 " 2, 99 " 3, 100 " 3 and 100 " 2%, respectively. and IC 50 values Ž12 " 3, 10 " 2, 13 " 3 and 14 " 3 nmolrl, respectively, n s 5..
4. Discussion The increase in blood pressure due to chronic endothelin-1 infusion was consistent with results of previous studies in normotensive rats ŽMortensen et al., 1990. and of studies showing the involvement of endothelin-1 in some types of hypertension, including angiotensin II ŽD’Uscio et al., 1997., L-NAME ŽLi et al., 1996. and deoxycorticosterone acetate ŽDOCA.-salt-induced hypertension ŽLariviere et al., 1993.. Chronic endothelin-1 did not change the vasoconstriction in response to phenylephrine. The acetylcholine-induced dilatation was increased in the aorta and unchanged in the mesenteric artery, despite an increase in systemic arterial pressure. The effect of the chronic endothelin-1 infusion was different in the 2 types of vessels. The absence of change in the amplitude of the response of mesenteric resistance arteries to both phenylephrine and acetylcholine suggests that other vascular beds may undergo a change in reactivity explaining the increase in blood pressure. The main effect of chronic endothelin-1 was a change in the type of vasoactive agent involved in acetylcholineinduced dilatation. In the aorta acetylcholine-induced-dilatation involved mainly NO but also constrictor cyclooxy-
genase productŽs. and some EDHF. The involvement of a vasoconstrictor cyclooxygenase product has been shown ŽKato et al., 1990., as was the involvement of EDHF in the aorta ŽFeletou and Vanhoutte, 1996.. The dilatation due to EDHF was blocked by KCl 15 mmolrl, a high enough concentration in the aorta Žtotal blockade of the dilatation remaining after L-NAME and indomethacin.. In the mesenteric artery, KCl 15 mmolrl blocked at least 90% of the dilatation remaining after L-NAME and indomethacin, suggesting that a fourth factor was involved or that EDHF was not totally blocked by KCl 15 mmolrl. Indeed, in mesenteric arteries concentrations of KCl higher than 15 mmolrl induced a contraction often higher than that required to test acetylcholine-induced dilatation. Chronic endothelin-1 markedly changed the type of agent involved in acetylcholine-induced dilatation in the aorta. The proportion of dilatation depending on the production of NO was decreased and dilator cyclooxygenase productŽs. were found instead of vasoconstrictor cyclooxygenase productŽs. after indomethacin. Thus, chronic endothelin-1 either increased the amount of vasodilator cyclooxygenase productŽs. or decreased the vasoconstrictor cyclooxygenase productŽs. Žor both.. In the mesenteric artery, the participation of cyclooxygenase productŽs. also increased after chronic endothelin-1. Endothelin-1 has previously been shown to increase vasodilator cyclooxygenase productŽs. in conductance arteries ŽMatsuda et al., 1993., which favors the hypothesis of an increased formation of vasodilator cyclooxygenase productŽs. on chronic endothelin-1 infusion. In mesenteric resistance arteries, the proportion of dilator agents found in response to acetylcholine was very different from that in the aorta. EDHF was the main vasodilator agent instead of NO. Indomethacin decreased slightly but significantly the response to acetylcholine, revealing the presence of vasodilator cyclooxygenase productŽs.. We have previously shown that both dilator and constrictor cyclooxygenase derivativeŽs. are produced by the rat mesenteric bed and that the balance between dilator and constrictor cyclooxygenase derivativeŽs. favors dilatation ŽMatrougui et al., 1997.. Thus the increased dilator response due to cyclooxygenase derivativeŽs. may reflect either an increased production of dilator cyclooxygenase derivativeŽs. or a decreased production of constrictor cyclooxygenase derivativeŽs.. In summary, chronic endothelin-1 modified the dilator capacity of aorta without affecting that of the mesenteric artery. Nevertheless, chronic endothelin-1 increased the participation of cyclooxygenase productŽs. in acetylcholine-induced dilatation in both the aorta and the mesenteric resistance arteries. Acknowledgements M.I. was a fellow of the ‘Fondation pour la Recherche ŽParis, France.. Medicale’ ´
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Lew, M.J., McPherson, A., 1996. Isolated tissues techniques. In: Garland, C.J., Angus, J.A. ŽEds.., Pharmacology of Vascular Smooth Muscle. Oxford Univ. Press, Oxford, pp. 25–41. Li, J.S., Deng, L.Y., Grove, K., Deschepper, C.F., Schiffrin, E.L., 1996. Comparison of effect of endothelin antagonism and angiotensin-converting enzyme inhibition on blood pressure and vascular structure in spontaneously hypertensive rats treated with N v-nitro-L-arginine methyl ester. Hypertension 28, 188–195. Magazine, H.I., Srivastava, K.D., 1996. Thrombin-induced vascular reactivity is modulated by ETB receptor-coupled nitric oxide release in rat aorta. Am. J. Physiol. 271, C923–C930. Matrougui, K., Maclouf, J., Levy, ´ B.I., Henrion, D., 1997. Impaired nitric oxide- and prostaglandin-mediated responses to flow in resistance arteries of hypertensive rats. Hypertension 30, 942–949. Matsuda, H., Eppu, S. or, Ohmori, F., Yamada, M., Miykatase, K., 1993. Involvement of cyclo-oxygenase-generated vasodilating eicosanoidŽs. in addition to nitric oxide in endothelin-1-induced endothelium-dependent vasorelaxation in guinea pig aorta. Heart Vessels 8, 121–128. Michaelis, L., Menten, T., 1913. Die kinetic der invertinwirkung. Biochem. Z. 49, 333–335. Mortensen, L.H., Pawloski, C.M., Kanaguy, N.L., Fink, G.D., 1990. Chronic hypertension produced by infusion of endothelin in rats. Hypertension 15, 729–735. Mulvany, M.J., Halpern, W., 1977. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circulation 41, 19–27. Nakashima, M., Vanhoutte, P.M., 1993. Endothelin-1 and -3 cause endothelium-dependent hyperpolarization in the rat mesenteric artery. Am. J. Physiol. 265, H2137–H2142. Oyekan, A., Balazy, M., Mc Giff, J.C., 1997. Renal oxygenases: differential contribution to vasoconstriction induced by endothelin-1 and angiotensin II. Am. J. Physiol. 273, R293–R299. Rabelink, T.J., Kaasjager, K.A., Stroes, E.S., Koomans, H.A., 1996. Endothelin in renal pathophysiology: from experimental to therapeutic application. Kidney Int. 50, 1827–1832. Sakai, S., Miyauchi, T., Sakurai, T., Kasuya, Y., Yamaguchi, I., Goto, K., Sugishata, Y., 1996. Endogenous endothelin-1 participates in the maintenance of cardiac function in rats with congestive heart failure. Circulation 93, 1214–1220. Sakuma, I., Asajima, H., Fukao, M., Tohse, N., Tamura, M., Kitabatake, A., 1993. Possible contribution of potassium channels to the endothelin-induced dilatation of rat coronary vascular beds. J. Cardiovasc. Pharmacol. 22, S232–S238. Sharma, A.C., Motew, S.J., Farias, S., Alden, K.J., Bosmann, H.B., Law, W.R., Fergusson, J.L., 1997. Sepsis alters myocardial and plasma concentrations of endothelin and nitric oxide in rats. J. Mol. Cell. Cardiol. 29, 1469–1475. Shetty, S.S., Okada, T., Webb, R.L., Delgrande, D., Lappe, R.W., 1993. Functionally distinct endothelin B receptors in vascular endothelium and smooth muscle. Biochem. Biophys. Res. Commun. 191, 459–465. Suzuki, N., Miyauchi, T., Tomobe, Y., Matsumoto, H., Oto, K., Masaki, T., Fujino, M., 1990. Plasma concentrations of endothelin-1 in spontaneously hypertensive rats and DOCA-salt hypertensive rats. Biochem. Biophys. Res. Commun. 167, 941–945. Yanagisawa, M., Kurihara, H., Kimura, S., Kasuya, Y.T., Miyauchi, Goto, K., Masaki, T., 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411–413. Zuccarello, M., Baccaleti, R., Tosun, M., Rapoport, R.M., 1996. Role of extracellular Ca2q in subarachnoid hemorrhage-induced spasm of the rabbit basilar artery. Stroke 27, 896–899.
Chronic endothelin-1-induced changes in vascular reactivity in rat resistance arteries and aorta Marc Iglarz, Bernard I. Levy, ´ Daniel Henrion
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(INSERM) U 141, IFR Circulation-Lariboisiere, Institut National de la Sante´ et de la Recherche Medicale Lariboisiere, ´ ` UniÕersite´ Paris VII, Hopital ˆ ` 41 bd de la Chapelle 75475 Paris, France Received 27 March 1998; revised 17 August 1998; accepted 18 August 1998
Abstract The role of endothelin-1 in vascular homeostasis is not yet clearly established. We investigated the responses to phenylephrine and acetylcholine in rat mesenteric resistance artery and aorta mounted in vitro in myographs after a 2-week treatment with endothelin-1 Ž5 pmol kgy1 miny1 , n s 8.. Systolic arterial blood pressure increased in endothelin-1-treated rats Ž171 " 7 mmHg vs. 196 " 6 mmHg, P - 0.05.. In the aorta, chronic endothelin-1 significantly increased the dilator response to acetylcholine Žmaximal dilatation: 76 " 3 vs. 86 " 3% in control, P - 0.05.. Acetylcholine-induced dilatation was decreased by nitric oxide ŽNO. synthase inhibition with N G-nitroL-arginine methyl ester ŽL-NAME 100 mmolrl. and partly restored by cyclooxygenases inhibition Žindomethacin, 10 mmolrl.. In endothelin-1-treated rats, L-NAME-sensitive acetylcholine dilatation was lower than in the control, but dilator cyclooxygenase productŽs. were found instead of constrictor cyclooxygenase productŽs.. In mesenteric resistance arteries chronic endothelin-1 increased the participation of cyclooxygenase products in acetylcholine-induced dilatation from 10 " 2 to 19 " 3%. In both types of arteries, phenylephrine-induced contraction was not affected by chronic endothelin-1. Thus chronic endothelin-1 increased the participation of dilator cyclooxygenase productŽs. in acetylcholine-induced dilatation in the aorta and the mesenteric resistance arteries. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Acetylcholine; Phenylephrine; Resistance artery; Aorta; Vascular reactivity; Indomethacin; N G -nitro-L-arginine methyl ester ŽL-NAME.; Endothelin; Hypertension
1. Introduction Endothelin-1 is a potent vasoconstrictor and mitogen produced by endothelial and vascular smooth muscle cells ŽYanagisawa et al., 1988.. This hormone exerts its biological effects via activation of specific endothelin-1 type A ŽETA . and type B ŽETB . receptors. Endothelin-1 type A receptors, which are expressed by vascular smooth muscle cells cause vasoconstriction ŽHaynes et al., 1995.. Endothelin-1 type B receptors expressed by vascular smooth muscle cells are also responsible for vasoconstriction and those expressed by endothelial cells can cause vasorelaxation ŽNakashima and Vanhoutte, 1993; Shetty et al., 1993.. The effects of endothelin-1 on vascular tone are different depending on species and vessel type. This reflects a difference in receptors distribution and a difference in the type of effectors produced upon endothelin-1 stimu) Corresponding author. Tel.: q33-1-4285-8672; Fax: q33-1-44631864; E-mail: [email protected]
lation. Endothelin-1-induced contraction may be mediated by cyclooxygenase ŽBarnett et al., 1994. or cytochrome P450 products ŽOyekan et al., 1997.. Endothelin-1-dependent vasorelaxation may also be mediated by one or several agents such as cyclooxygenase productŽs. ŽLal et al., 1996, Matsuda et al., 1993., nitric oxide ŽNO. ŽMagazine and Srivastava, 1996. and endothelium-derived hyperpolarizing factorŽs. ŽEDHF. ŽSakuma et al., 1993.. Endothelin-1 might be involved in several pathological situations such as heart failure ŽSakai et al., 1996., preeclampsia ŽGreer et al., 1991., renal dysfunction ŽRabelink et al., 1996; Karam et al., 1996., sepsis ŽSharma et al., 1997., atherosclerosis ŽHasdai et al., 1997., cerebral vasospasm ŽCosentino and Katusic, 1994; Zuccarello et al., 1996., in effects of aging ŽBattistelli et al., 1996. and in hypertension ŽSuzuki et al., 1990; Kohno et al., 1991; Lariviere et al., 1993; Li et al., 1996. or effects of chronic administration of angiotensin II ŽD’Uscio et al., 1997.. No study, however, has yet defined the changes in vascular reactivity due to chronic elevation of endothelin-1 in either
0014-2999r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 1 4 - 2 9 9 9 Ž 9 8 . 0 0 6 1 6 - 5
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large or in resistance vessels. Chronic infusion of endothelin-1 increases blood pressure ŽMortensen et al., 1990. and physiological concentrations of endothelin-1 raise the agonist-induced tone ŽHenrion and Laher, 1993.. Our working hypothesis was that chronic administration of endothelin-1 should change the vascular reactivity to both vasodilator and vasoconstrictor agents as endothelin-1 is involved in both dilator and constrictor processes Žsee above.. We investigated the impact of chronic endothelin-1 treatment on the vascular reactivity of large Žaorta. and small resistance arteries Žmesenteric. to phenylephrine and acetylcholine.
2. Materials and methods 2.1. Animal preparation Fifteen male Wistar–Kyoto rats ŽIffa-Credo, Lyon, France., 10-weeks-old and weighing 250 g, were randomly allocated to a control group Žfed with standard chow and tap water, n s 8. or to a group receiving endothelin-1 subcutaneously Ž5 pmol kgy1 miny1, osmotic minipumps Alzet, model 2002, Palo Alto, CA, USA, n s 7. for 2 weeks. After 2 weeks, systolic blood pressure was monitored by the tail cuff method ŽBP recorder 8006, Ugo Basile, Comerio, Italy.. The thoracic aorta and a section of the mesenteric bed were dissected out and immersed in ice-cold physiological salt solution Žcomposition described below.. The procedure followed for the care and killing of the study animals was in accordance with the European Community standards on the care and use of laboratory animals ŽMinistere ` de l’Agriculture, France, authorisation no. 00577.. 2.2. Isolated ring segments of aorta Ring segments of aorta, cleaned of fat and connective tissues, 3 mm in length, were mounted between two stainless-steel wires in 3-ml organ baths containing physiological salt solution of the following composition Žin mmolrl.: 135.0 NaCl, 15.0 NaHCO 3 , 4.6 KCl, 1.5 CaCl 2 , 1.2 MgSO4 , and 11.0 glucose. The solution was bubbled with 95% O 2 and 5% O 2 and the pH was 7.4. Physiological salt solutions containing Kq 15 or 60 mmolrl were prepared by equimolar substitution of KCl for NaCl. One wire was attached to a fixed support while the second wire was connected to a moveable holder supporting a tension transducer ŽGrass FT.03, Quincy, MA, USA. so that isometric force measurements could be collected by a Biopac data acquisition system ŽBiopac MP 100, LaJolla, CA, USA. and recorded by a computer ŽApple computers, Cupertino, CA, USA., using the Acknowledge w data acquisition and analysis software ŽBiopac, LaJolla, CA, USA.. The arterial segments were allowed to recover for 30 min. during which time the physiological salt solution
was replaced at 15-min intervals. Following this recovery period, stepwise increases in tension were applied to each segment and the response to KCl 60 mmolrl was determined at each level of tension. The optimal tension was determined as the tension corresponding to the optimal response to KCl. The average tension needed was 2 g, irrespective of the treatment Ž2.04 " 0.07 g, n s 32 segments in control and 1.93 " 0.09 g, n s 28 segments in endothelin-1-treated rats.. Segments of aorta were allowed to equilibrate for an additional 90 min. Four segments of aorta were isolated per rat and a concentration-response curve for one of the drugs described below was obtained. At the end of the experimental protocol aorta ring segments were blotted dry and weighed. Concentration–response curves for phenylephrine Ž1 nmolrl to 100 mmolrl. were obtained by cumulative addition of phenylephrine to the bath solution. The data are N force per g tissue. Concentration–response curves for acetylcholine Ž1 nmolrl to 10 mmolrl. were obtained by cumulative addition of acetylcholine to the physiological salt solution after preconstriction of the arterial segments with a concentration of phenylephrine Ž30 to 300 nmolrl. sufficient to reach approximately 70% of the tissue maximum as determined with Kq Ž60 mmolrl.. Data are expressed as % dilatation of phenylephrine-induced preconstriction. Concentration–response curves for phenylephrine and acetylcholine were repeated after incubation of the segments of aorta for 30 min. with the NO synthase inhibitor, N G -nitro-L-arginine methyl ester ŽL-NAME. 100 mmolrl, or L-NAME 100 mmolrlq the non-selective cyclooxygenase inhibitor indomethacin 10 mmolrl, or L-NAME 100 mmolrl q indomethacin 10 mmolrl q KCl 15 mmolrl. As NO synthesis blockade with L-NAME may not be complete ŽGriffith and Stuehr, 1995. we used a concentration 10 times higher than that in previous studies in which L-NAME has been shown to block NO-dependent responses ŽHenrion et al., 1997a,b. and one with an effect equivalent to that of another blocker ŽDowell et al., 1996.. No drug was added in the fourth segment of aorta before repetition of the concentration–response curve for phenylephrine and acetylcholine. This segment of aorta served as a time control. In another series of experiments, indomethacin 10 mmolrl or KCl 15 mmolrl was added to the bath solution in the absence of other substances. 2.3. Isolated mesenteric resistance arteries Segments of second-order mesenteric artery Žapprox. 200 mm external diameter. were trimmed free of fat and adhering connective tissue, and mounted in a myograph according to the technique of Mulvany and Halpern Ž1977.. Isometric tension was recorded and collected with a Biopac data acquisition system ŽBiopac MP 100. and recorded by a computer ŽApple computers. using the Acknowledge w
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reach approximately 70% of the tissue maximum as determined with Kq Ž60 mmolrl.. The data are expressed as % dilatation of phenylephrine-induced preconstriction. The tissues were allowed to equilibrate for 15 min between responses. Concentration–response curves to phenylephrine and acetylcholine were repeated after successive incubation of the segments of mesenteric arteries for 20 min with LNAME Ž100 mmolrl., indomethacin Ž10 mmolrl., and then KCl 15 mmolrl. In a separate series of experiments, 4 successive concentration–response curves to phenylephrine and acetylcholine were made with segments of mesenteric artery without the successive addition of LNAME Ž100 mmolrl., indomethacin Ž10 mmolrl., and KCl 15 mmolrl. This group of experiments served as a time control. In separate series of experiments indo-
Fig. 1. Concentration-dependent contraction in response to phenylephrine in rat aorta Župper panel. and mesenteric resistance artery segments Žlower panel.. Rats were treated for 2 weeks with endothelin-1 Ž5 pmol kgy1 miny1 , open circles. or with the solvent Žopen squares.. Data are given as the means"S.E.M. Two-factor ANOVA for consecutive measurements, endothelin-1 vs. control: no significant difference.
data acquisition and analysis software ŽBiopac.. Segments were allowed to equilibrate for 30 min in physiological salt solution, maintained at 378C and gassed with 95% O 2 and 5% CO 2 . The physiological salt solution was replaced every 15 min. Each vessel was placed under optimal stretch, equivalent to an in vivo arterial blood pressure of approximately 100 mmHg ŽMulvany and Halpern, 1977; Lew and McPherson, 1996.. The vessels were allowed to equilibrate for 30 min. Tissue contractility was then assessed by exposure to Kq Ž60 mmolrl.. Subsequently, cumulative concentration–response curves were obtained for phenylephrine Ž10 nmolrl to 100 mmolrl.. The data are expressed as mN force per mm vessel length. Acetylcholine concentration–response curves Ž1 nmolrl to 10 mmolrl. were obtained after a preconstriction of the mesenteric rings with a concentration of phenylephrine Ž10 to 100 nmolrl. sufficient to
Fig. 2. Concentration-dependent dilatation in response to acetylcholine in rat aorta Župper panel. and mesenteric resistance artery segments Žlower panel.. Rats were treated for 2 weeks with endothelin-1 Ž5 pmol kgy1 miny1 , open circles. or with the solvent Žopen squares.. Data are given as the means"S.E.M. aP - 0.05, two-factor ANOVA, endothelin-1 vs. control. ) P - 0.05, one-way ANOVA, endothelin-1 vs. control.
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where E is the contraction, Emax is the maximum contraction, C is the concentration, EC is the EC 50 ŽIC 50 for relaxation. Žconcentration of agonist required to induce half the maximum response. and m is the Hill coefficient. Comparisons between groups were made using a one factor analysis of variance ŽANOVA. followed by Dunnett’s t-test or by two-factor ANOVA for repeated measures to compare the concentration–response curves Žtreated animals versus controls or relaxation pathway inhibitors versus control.. A probability level of P - 0.05 was considered significant.
Fig. 3. Effect of the consecutive addition of L-NAME Ž100 mmolrl., indomethacin Ž10 mmolrl. and KCl Ž15 mmolrl. on the concentrationdependent dilatation in response to acetylcholine in aorta ring segments isolated from rats treated for 2 weeks with endothelin-1 Ž5 pmol kgy1 miny1 , lower panel. or with the solvent Župper panel.. Data are given as the means"S.E.M. Groups were control Žopen squares., L-NAME Žclosed squares., indomethacin Žclosed circles. and KCl Žopen circles.. ) P 0.05, one-way ANOVA, L-NAME versus control. §P - 0.05, one-way ANOVA, L-NAMEqindomethacin vs. L-NAME. aP - 0.05, one-way ANOVA, L-NAMEqindomethacinqKCl vs. L-NAMEqindomethacin. Two-factor ANOVA: L-NAME vs. control Ž P - 0.05, lower and upper panel., L-NAMEqindomethacin vs. control or L-NAME Ž P - 0.05, lower and upper panel. and L-NAMEqindomethacinqKCl vs. L-NAMEq indomethacin Ž P - 0.05, upper panel only..
methacin 10 mmolrl or KCl 15 mmolrl was added to the bath solution in the absence of other substances. 2.4. Statistical analysis The results are expressed as means " S.E.M. EC 50 ŽIC 50 for relaxation. and Emax were calculated individually for each concentration–response curve using the equation ŽMichaelis and Menten, 1913.: E s Ž Emax = C m . r Ž EC m q C m .
Fig. 4. Effect of the addition of L-NAME Ž100 mmolrl., indomethacin Ž10 mmolrl. and KCl Ž15 mmolrl. on the concentration-dependent dilatation in response to acetylcholine in mesenteric artery segments isolated from rats treated for 2 weeks with endothelin-1 Ž5 pmol kgy1 miny1 , lower panel. or with the solvent Župper panel.. Groups were control Žopen squares., L-NAME Žclosed squares., indomethacin Žclosed circles. and KCl Žopen circles.. Data are given as the means"S.E.M. ) P - 0.05, one-way ANOVA, L-NAME vs. control. §P - 0.05, one-way ANOVA, L-NAMEqindomethacin vs. L-NAME. aP - 0.05, one-way ANOVA, L-NAMEqindomethacinqKCl vs. L-NAMEqindomethacin. Two-factor ANOVA Ž P - 0.05, lower and upper panel.: L-NAME vs. control, L-NAMEqindomethacin versus control or L-NAME and LNAMEqindomethacinqKCl vs. L-NAMEqindomethacin.
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2.5. Drugs
3.3. Acetylcholine-induced dilatation
Endothelin-1, acetylcholine, phenylephrine, L-NAME and indomethacin were purchased from Sigma ŽSt. Louis, MO, USA.. The other reagents were obtained from Prolabo ŽParis, France..
Acetylcholine Ž1 nmolrl to 10 mmolrl. induced a concentration-dependent dilatation of aortic and mesenteric artery ring segments precontracted with phenylephrine ŽFig. 2.. Precontraction with phenylephrine was equivalent in all groups. Chronic endothelin-1 significantly increased acetylcholine-induced dilatation in the aorta ŽFig. 2; Emax s 76 " 3 vs. 86 " 3% relaxation, P - 0.05; no significant change in EC 50 : 29 " 7 nmolrl vs. 48 " 9 nmolrl.. The greatest increase in acetylcholine-induced dilatation was observed when acetylcholine was 30 nmolrl Ž42% increase in dilatation, Fig. 2.. On the other hand, chronic endothelin-1 did not significantly affect acetylcholine-induced dilatation in mesenteric resistance arteries ŽFig. 2.. The addition of L-NAME Ž100 mmolrl., indomethacin Ž10 mmolrl. or KCl Ž15 mmolrl. significantly attenuated acetylcholine-induced dilatation in aorta ŽFig. 3. and in mesenteric artery ring segments ŽFig. 4.. In the aorta, L-NAME Ž100 mmolrl. induced a significant attenuation of acetylcholine-induced dilatation ŽFig. 3.. Chronic endothelin-1 induced a significant change in L-NAME Ž100 mmolrl.-induced attenuation of acetylcholine dilatation ŽFigs. 3 and 5.. In the aorta, indomethacin Ž10 mmolrl., in addition to L-NAME, significantly increased acetylcholine-induced dilatation, reflecting the participation of vasoconstrictor cyclooxygenase productŽs. in the response to acetylcholine Žrepresenting 24 " 5% of the total response to acetylcholine, Fig. 5.. In contrast, in the aorta of rats treated chronically with endothelin-1, indomethacin Ž10 mmolrl., in addition to LNAME, further decreased acetylcholine-induced dilatation,
3. Results 3.1. Blood pressure A 2-week treatment with endothelin-1 significantly increased systolic arterial pressure Ž196 " 6 mm Hg vs. 171 " 7 in the control group, P - 0.05.. 3.2. Phenylephrine-induced contraction Phenylephrine induced a concentration-dependent contraction in aorta and mesenteric resistance arterial segments ŽFig. 1.. Chronic endothelin-1 had no significant effect on phenylephrine-induced contraction in either aorta or mesenteric arterial segments ŽFig. 1.. The consecutive addition of L-NAME Ž100 mM. and indomethacin Ž10 mM. did not significantly affect the response to phenylephrine in the aorta or in the mesenteric artery Ždata not shown.. KCl Ž15 mM. significantly increased Ž P - 0.005. the response to phenylephrine ŽEC 50 from 43 " 6 to 7.2 " 1.1 nmolrl in the aorta and from 1.1 " 0.16 to 0.18 " 0.05 mmolrl in the mesenteric artery; no effect of chronic endothelin-1..
Fig. 5. Effect of chronic endothelin-1 treatment on the distribution of vasoactive agents in the dilator response to acetylcholine in the aorta and in the mesenteric artery. Bars represent the mean percentage Ž"S.E.M.. of each agent calculated by measuring the areas under the curve ŽFig. 3Fig. 4. of each concentration–response curve to acetylcholine obtained after the successive addition of the inhibitors: L-NAME, indomethacin and then KCl. Data are given as the means and S.E.M. Žnot presented. are less than 10% of means. ) P - 0.05, one-way ANOVA, chronic endothelin-1 vs. control.
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reflecting the participation of vasodilator cyclooxygenase productŽs. in acetylcholine-induced dilatation Žrepresenting 40 " 6% of the total response to acetylcholine, Figs. 3 and 5.. The further addition of KCl Ž15 mmolrl., after L-NAME and indomethacin, abolished the residual acetylcholine-induced dilatation of aortic rings ŽFig. 3.. The effect of KCl Ž15 mmolrl. was significantly decreased after chronic endothelin-1 ŽFig. 5.. The effect of indomethacin or KCl Ž15 mmolrl. was identical to that described above when indomethacin or KCl Ž15 mmolrl. was used alone Ždata not shown.. In mesenteric resistance artery segments, the successive addition of L-NAME Ž100 mmolrl., indomethacin Ž10 mmolrl. and KCl Ž15 mmolrl. significantly attenuated acetylcholine-induced dilatation and allowed the differentiation of 3 distinct dilator pathways: NO, suppressed by L-NAME, cyclooxygenase productŽs., suppressed by indomethacin and EDHF, suppressed by KCl ŽFigs. 4 and 5.. Chronic endothelin-1 increased the participation of cyclooxygenase products in acetylcholine-induced dilatation from 10 " 2 to 19 " 3% ŽFig. 5.. In a separate group of experiments 4 successive concentration-response curves for acetylcholine were obtained with mesenteric resistance arteries, without the addition of L-NAME, indomethacin or KCl, in order to test the effect of time on the responses. The 4 successive curves had similar Emax Ž98 " 2, 99 " 3, 100 " 3 and 100 " 2%, respectively. and IC 50 values Ž12 " 3, 10 " 2, 13 " 3 and 14 " 3 nmolrl, respectively, n s 5..
4. Discussion The increase in blood pressure due to chronic endothelin-1 infusion was consistent with results of previous studies in normotensive rats ŽMortensen et al., 1990. and of studies showing the involvement of endothelin-1 in some types of hypertension, including angiotensin II ŽD’Uscio et al., 1997., L-NAME ŽLi et al., 1996. and deoxycorticosterone acetate ŽDOCA.-salt-induced hypertension ŽLariviere et al., 1993.. Chronic endothelin-1 did not change the vasoconstriction in response to phenylephrine. The acetylcholine-induced dilatation was increased in the aorta and unchanged in the mesenteric artery, despite an increase in systemic arterial pressure. The effect of the chronic endothelin-1 infusion was different in the 2 types of vessels. The absence of change in the amplitude of the response of mesenteric resistance arteries to both phenylephrine and acetylcholine suggests that other vascular beds may undergo a change in reactivity explaining the increase in blood pressure. The main effect of chronic endothelin-1 was a change in the type of vasoactive agent involved in acetylcholineinduced dilatation. In the aorta acetylcholine-induced-dilatation involved mainly NO but also constrictor cyclooxy-
genase productŽs. and some EDHF. The involvement of a vasoconstrictor cyclooxygenase product has been shown ŽKato et al., 1990., as was the involvement of EDHF in the aorta ŽFeletou and Vanhoutte, 1996.. The dilatation due to EDHF was blocked by KCl 15 mmolrl, a high enough concentration in the aorta Žtotal blockade of the dilatation remaining after L-NAME and indomethacin.. In the mesenteric artery, KCl 15 mmolrl blocked at least 90% of the dilatation remaining after L-NAME and indomethacin, suggesting that a fourth factor was involved or that EDHF was not totally blocked by KCl 15 mmolrl. Indeed, in mesenteric arteries concentrations of KCl higher than 15 mmolrl induced a contraction often higher than that required to test acetylcholine-induced dilatation. Chronic endothelin-1 markedly changed the type of agent involved in acetylcholine-induced dilatation in the aorta. The proportion of dilatation depending on the production of NO was decreased and dilator cyclooxygenase productŽs. were found instead of vasoconstrictor cyclooxygenase productŽs. after indomethacin. Thus, chronic endothelin-1 either increased the amount of vasodilator cyclooxygenase productŽs. or decreased the vasoconstrictor cyclooxygenase productŽs. Žor both.. In the mesenteric artery, the participation of cyclooxygenase productŽs. also increased after chronic endothelin-1. Endothelin-1 has previously been shown to increase vasodilator cyclooxygenase productŽs. in conductance arteries ŽMatsuda et al., 1993., which favors the hypothesis of an increased formation of vasodilator cyclooxygenase productŽs. on chronic endothelin-1 infusion. In mesenteric resistance arteries, the proportion of dilator agents found in response to acetylcholine was very different from that in the aorta. EDHF was the main vasodilator agent instead of NO. Indomethacin decreased slightly but significantly the response to acetylcholine, revealing the presence of vasodilator cyclooxygenase productŽs.. We have previously shown that both dilator and constrictor cyclooxygenase derivativeŽs. are produced by the rat mesenteric bed and that the balance between dilator and constrictor cyclooxygenase derivativeŽs. favors dilatation ŽMatrougui et al., 1997.. Thus the increased dilator response due to cyclooxygenase derivativeŽs. may reflect either an increased production of dilator cyclooxygenase derivativeŽs. or a decreased production of constrictor cyclooxygenase derivativeŽs.. In summary, chronic endothelin-1 modified the dilator capacity of aorta without affecting that of the mesenteric artery. Nevertheless, chronic endothelin-1 increased the participation of cyclooxygenase productŽs. in acetylcholine-induced dilatation in both the aorta and the mesenteric resistance arteries. Acknowledgements M.I. was a fellow of the ‘Fondation pour la Recherche ŽParis, France.. Medicale’ ´
M. Iglarz et al.r European Journal of Pharmacology 359 (1998) 69–75
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