Acute effect of IT@-estradiol on rabbit coronary contractile responses to endothelin-1 CANWEN
JIANG,
PHILIP
M. SARREL,
PHILIP
A. POOLE-WILSON,
Department of Cardiac Medicine, National Heart and Lung Institute, London SW3 6LY, United Kingdom Jiang, Canwen, Philip M. Sarrel, Philip A. Poole-Wilson, and Peter Collins. Acute effect of 17@-estradiolon rabbit coronary artery contractile responsesto endothelin-1. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H271-H275, 1992.-We assessed the acute effect of 17@-estradiolon coronary artery constrictor responsesto endothelin-1. 17P-Estradiol significantly shifted endothelin-1, calcium, or BAY K 8644 concentration-dependent contraction curves to the right in endotheliurn-denuded coronary arteries isolated from nonpregnant female rabbits. The -log 50% effective dose(ED& of calcium in high KC1 medium (100mM) was3.8 t 0.11 in control and 3.2 & 0.1 and 2.8 t 0.12 after incubation with 17@-estradiol(1 and 10 PM, respectively). The -log EDs0 of BAY K 8644 (KC1 15 mM) was 7.8 * 0.1 in control and 7.4 t 0.08 and 7.2 t 0.05 in the presenceof 1’7@-estradiol (1 and 10 PM, respectively). The -log EDs0 of endothelin-1 was 9.2 t 0.08 in control and 8.8 t 0.1, 8.4 t 0.07, and 8.1 t 0.12 after incubation with 17@-estradiol (3, 10, and 30 PM, respectively). Similar results were obtained from coronary arteriesof malerabbits. Theseincreasesof -log EDs0 values were significant (P < 0.05 or 0.01). 17P-Estradiol and verapamil induced dose-dependentrelaxation in both endothelium-intact or -denudedcoronary arteries submaximally precontracted by endothelin-1. NG-monomethyl+arginine had no effect on relaxation induced by 17P-estradiol, whereasit eliminated relaxation induced by acetylcholine in rings with an intact endothelium. These data suggestthat 17@estradiol attenuates the rabbit coronary artery contraction induced by endothelin- 1 via an endothelium-independent mechanism, possibly by affecting calcium influx. endothelium; endothelium-derived relaxing factor; verapamil; relaxation; calcium influx ENDOTHELIUM PLAYS an important role in the regulation of coronary arterial tone by releasing both endothelium-derived relaxing and contracting factors. Endothelin-1 is a vasoconstrictor peptide isolated from the supernatant of cultured porcine endothelial cells (31, 32). It induces potent contraction in isolated coronary arteries (12). Endothelium-derived relaxing factor (EDRF) has been shown to inhibit smooth muscle contraction induced by endothelin-1 in canine arteries and veins (19) and in human arteries (15). In certain pathophysiological conditions such as atherosclerosis, endothelium-dependent vasodilation is impaired and endothelium-dependent contraction may predominate (14). Exogenous estrogen administration to guinea pigs induces similar hemodynamic changes to those seen in pregnancy (4, 10). Endothelium-dependent vasodilation is impaired in ovariectomized rabbits and spontaneously hypertensive rats, but this is partially restored by chronic estrogen administration in these animals (6, 18, 30). Intracoronary infusion of acetylcholine induces coronary artery contraction in ovariectomized and atherosclerotic monkeys but relaxation in animals treated chronically with estrogen (29). Endothelin-1 -induced contraction may occur in coronary atherosclerosis and 0363-6135/92
artery AND PETER
COLLINS
University of London,
play a role in the pathophysiology of coronary artery spasm and myocardial ischemia (11, 14, 28). However, the effect of UP-estradiol on coronary artery contraction induced by endothelin-1 is not known. In this study, we have investigated the acute effect of 17&estradiol on contractile responses to endothelin-1 in isolated coronary arterial preparations of male or nonpregnant female rabbits. MATERIALS AND METHODS Preparation of rabbit coronary arteries. Adult male or nonpregnant female New Zealand White rabbits (2.5-3 kg) were anesthetizedby an injection of pentobarbital sodium(60 mg/kg) into the ear vein after an injection of heparin (150 U/kg) into the ear vein. The hearts were removed and immediately immersedin ice-cold modified Krebs solution. The left anterior descendingcoronary artery was dissected free of connective tissue. Rings (2-3 mm length) were prepared, and in alternate rings, endothelium was removed by gently rubbing the intimal surfacewith a woodenprobe. The presenceor absenceof endothelium wasverified by the relaxation of the rings in responseto substanceP (0.3, 1, 3, and 10 nM). Measurement of arterial tension. Coronary artery rings with or without endothelium were suspendedhorizontally between two stainlesssteel hooksin individual organ baths containing 7 ml modified Krebs solution at 37’C, bubbled with 95% 02-5% C02, for measurementof isometric tension. The compositionof modified Krebs solution was (in mM) 118.3NaCl, 4.7 KCl, 2.5 CaC1,, 1.2 MgSO*, 1.2 K2P04, and 11.1 glucose.Rings were equilibrated for 90 min under a resting tension of 1 g before drugswere added.Except for one group, in which indomethacin was omitted, all experiments were performed in rings with or without endothelium in the presenceof indomethacin (10 PM). Effect of 17p-estrudiol or verapamil on endothelin-1 -induced contraction. Endothelin-1 concentration-dependentcontraction
curves in modified Krebs solution wereperformed in rings without endothelium in the absenceof l’?P-estradiol or verapamil. Becausethe tension after the contraction induced by endothelin-1 did not return to basal levels after repeatedwashout, the concentration-dependent responsecurves in the presenceand the absenceof 17P-estradiol or verapamil could not be performed in the samerings. In different tissues,after incubation with 17&estradiol (3, 10, and 30 PM; dissolved in ethanol), verapamil(O.1, 1, and 10PM; dissolvedin ethanol), or the same concentration of the solvent ethanol (1:2,000 vol) for 20 min, endothelin-1 concentration-dependent contraction curves were independently obtained. In the other group, the relaxing responsesto 17@-estradiol(1, 3, 10, 30, and 100 PM), verapamil (0.1, 0.3, 1, 3, 10, and 30 PM), or the sameconcentration of ethanol were measuredin rings after being submaximally contracted by endothelin-1 (3 nM). The relaxing responsesto 17@estradiol (1, 3, 10, 30, and 100PM) were also measuredin one group of endothelium-intact rings contracted with endothelin-1 (3 nM) in the absenceof indomethacin. iVG-monomethyl+ arginine (L-NMMA, 30 and 100 PM) was used to assessthe EDRF-dependent component of the relaxation induced by 17@estradiol or verapamil. In addition, the effects of L-arginine or D-arginine (1 mM) on the L-NMMA-induced inhibition of the
$2.00 Copyright 0 1992 the American Physiological
Society
H271
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H272
l’I@ESTRADIOL
ATTENUATES
ENDOTHELIN-INDUCED
CONTRACTION
relaxing responses to acetylcholine were measured. Effects of 17P-estradiol on contraction induced by calcium and BAY K 8644. Endothelium-denuded coronary arteries from
nonpregnant female rabbits were incubated in calcium-free solution containing 0.5 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) for 10 min. Calcium concentration-dependent contraction curves were then performed in K+-depolarization medium (KC1 100mM). The rings were equilibrated in modified Krebs solution for 20 min before being incubated in calcium-free solution containing 0.5 mM EGTA for another 10 min. These rings were incubated with 17@-estradiol(1 and 10 PM) or the sameconcentration of the solvent ethanol before the calcium concentration-dependent contraction curves. In another group of rings from female rabbits, constrictor responsesto BAY K 8644 (0.01, 0.03, 0.1, 0.3, and 1 PM plus 15 mM KCl) in the absenceor presenceof I?‘@-estradiol(1 and 10 PM) were measured. Drugs. 17@-Estradiol,verapamil, endothelin-1, acetylcholine, L-arginine, D-arginine, and indomethacin were purchasedfrom Sigma Chemical (Poole, Dorset, UK). BAY K 8644 was obtained from Calbiochem(La Jolla, CA). L-NMMA (acetatesalt) was a generousgift from Dr. S. Moncada of Wellcome Laboratories (Beckenham, Kent, UK). Statistical analyses. Values are expressedas means k SE. The relaxation is expressedaspercentageof contraction. Data wereanalyzed by the analysisof variance, followed by NewmanKeuls test. P < 0.05 was consideredto be a statistically significant difference. The number of animals is indicated by n. RESULTS
Endothelin-1 -induced contraction in rabbit coronary arteries. Endothelin- 1 induced slowly developing concen-
tration-dependent contraction in isolated rabbit coronary arterial rings. The -log 50% effective dose (ED& values were 9.3 t 0.05 and 9.3 t 0.04, respectively, in endothelium-intact or -denuded coronary arterial rings from male rabbits (n = 6; P > 0.05). In nonpregnant female preparations (n = 6), the -log EDs0 values were 9.3 t 0.07 and 9.2 + 0.08, respectively, in endothelium-intact or -denuded arterial rings (P > 0.05). Endothelin-1 (3 nM) induced comparable submaximal contraction in endothelium-intact or -denuded rings of male rabbits (1.0 t 0.2 and 1.1 t 0.1 g, respectively, P > 0.05). Effect of 1 ?P-estradiol on endothelin-1 -induced contraction. After 20-min incubation, 17P-estradiol produced
a concentration-dependent antagonism of endothelin- linduced contraction in both male and female preparations. The same concentration of the solvent ethanol had no significant effect on this contraction. The endothelin-1 -induced concentration-dependent contraction curves were shifted to the right in a nonparallel fashion. In endothelium-denuded rings of male rabbits, the -log EDs0 values of endothelin-1 were increased to 8.8 t 0.09, 8.3 t 0.11, and 8.1 t 0.07 (P < 0.05 or 0.01 in comparison with control) after incubation with 17@-estradiol (3, 10, and 30 PM, respectively). After incubation with 17@estradiol (10 or 30 p M), the maximal contractile responses to endothelin-1 were suppressed by 8 and 2l%, respectively (Fig. 1). Similar results were obtained in endothelium-denuded coronary arteries from nonpregnant female rabbits (n = 5) in which the -log EDs0 values were 8.8 t 0.1, 8.4 t 0.07, and 8.1 t 0.12 (P < 0.05 or 0.01 in comparison with control) after incubation with 17@-estradiol (3, 10, and 30 PM, respectively). UP-Estradiol in-
10
9.5 9 Endothelin-
a.5 a 7.5 1 concentration
6
Fig. 1. Effect of UP-estradiol (1, 10, and 30 PM) on concentrationdependent contractile responses to endothelin-1 in male rabbit coronary arteries without endothelium. Data shown are means t SE in g (n = 6). Experiments were performed in presence of indomethacin (10 PM). Circles, squares, triangles, and diamonds indicate control, 0.1, 1, and 10 PM 17&estradiol, respectively.
duced relaxation of endothelin- 1 (3 nM) -precontracted endothelium-intact or -denuded rings of male rabbits. No differences were observed between rings with and without endothelium (Fig. 2). Similar results were obtained in the presence or absence of indomethacin (10 PM; data not shown, n = 6). L-NMMA (30 and 100 PM) had no inhibitory effect on the 17p-estradiol-induced relaxation (data not shown, n = 6). In contrast, acetylcholine caused concentration-dependent relaxation of endothelin-l-precontracted endothelium-intact arteries. The relaxation was completely abolished by the removal of the endothelium. L-NMMA inhibited this relaxation in a concentrationdependent manner. L-arginine but not D-arginine overcame the inhibition induced by L-NMMA (data not shown, n = 6). Effect traction.
of verapamil
on endothelin-1 -induced
con-
Verapamil significantly inhibited concentration-dependent constrictor responses to endothelin-1 in a manner similar to UP-estradiol in endothelium-denuded rings of male rabbits. The concentration-dependent contraction curves were shifted to the right in a nonparallel fashion. The -log EDs0 values were increased to 8.4 t 0.03, 7.8 t 0.11, and 7.4 t 0.06 (P c 0.01 in comparison with control) after incubation with verapamil (0.1, 1, and 100 -
a0 -
20 -
Fig. 2. Relaxing effect of 17/3-estradiol on endothelin-1 (3 nM)-precontracted rabbit coronary arteries with (circles) and without (squares) endothelium. Submaximal contractions induced by endothelin-1 were 1.0 t 0.2 and 1.1 t 0.1 g in endothelium-intact and -denuded rings, respectively. Data shown are percentage relaxation means t SE (n = 6, all P > 0.05). Experiments were performed in presence of indomethacin (10 PM).
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17@-ESTRADIOL
ATTENUATES
ENDOTHELIN-INDUCED
10 PM, respectively) compared with 9.3 t 0.03 in control. However, the maximal constrictor responses to endothelin-1 were suppressed by 21 and 41% after incubation with verapamil (3 and 10 PM, respectively). This antagonistic effect of verapamil was greater than that of 17@estradiol (Fig. 3). Verapamil(O.3,1,3,10, and 30 PM) also induced relaxation of endothelin- 1-precontracted endothelium-intact or -denuded rings of male rabbits (Fig. 4). No statistically significant differences were seen between rings with and without endothelium (P > 0.05). L-NMMA (30 and 100 PM) had no inhibitory effect on verapamil-induced relaxation (data not shown, n = 6). Inhibitory effect of 17@estradiol on calcium-induced contraction. The calcium concentration-dependent con-
traction curves in K+-depolarization medium were shifted to the right after incubation with 17P-estradiol in endothelium-denuded rings of nonpregnant female rabbits. The -log 50% effective concentration (EC,,) was increased from 3.8 t 0.11 in control to 3.2 t 0.1 and 2.8 t 0.12 (P < 0.05 and 0.01, respectively) after incubation with 17P-estradiol (1 and 10 PM, respectively). Maximal contraction was reduced by 21 t 4.4 and 34 t 5.4%, respectively (Fig. 5).
H273
CONTRACTION
120 100 1
g 8o .g 60: $ 40 20 O7
6 5.5 concentration
VeradLnil
5 (-log
4.5 M)
Fig. 4. Effect of verapamil on endothelin-1 (3 nM)-precontracted rabbit coronary arteries with (triangles) and without (squares) endothelium. Submaximal contractions induced by endothelin-1 were 1.1 t 0.2 and 1.1 k 0.2 g in endothelium-intact and -denuded rings, respectively. Data shown are percentage relaxation means t SE (n = 6, P > 0.05).
Inhibitory effect of 17@-estradiol on BAY K 8644-induced contraction. BAY K 8644 (0.01, 0.03, and 0.1 PM
plus 15 mM KCl) induced concentration-dependent contraction in endothelium-denuded rings of nonpregnant female rabbits. Higher concentrations of BAY K 8644 (0.3 and 1 PM) did not cause further contraction and induced slight reduction of contraction evoked by lower concentrations in some preparations. In the presence of 17@-estradiol (1 and 10 PM), BAY K 8644-induced contraction was markedly reduced (P < 0.05 or 0.01, respectively, Fig. 6). The -log ECSO values were 7.8 t 0.1 in control and 7.4 t 0.08 and 7.2 t 0.05 in the presence of 17P-estradiol (1 and 10 PM, respectively).
0 ’ 4
3.5 Calcium
3 2.5 Concentrations
2 (-log
1.5 M)
Fig. 5. Effect of 17P-estradiol (1 and 10 PM, squares and triangles, respectively) on calcium concentration-dependent contraction (control, circles) in high KC1 (100 mM) medium, in endothelium-denuded coronary arteries of female rabbits. Data are expressed as percentage contraction (means k SE, n = 5) of maximal contraction induced by calcium in control condition. Experiments were performed in presence of indomethacin (10 PM).
DISCUSSION
We have demonstrated that endothelin-1 is a potent constrictor of the rabbit coronary artery. Our results are consistent with a previous report in the porcine coronary artery (12). Activation of voltage-dependent calcium channels is thought to be a part of the mechanism of endothelin-1 contraction. This involves binding to spe2.0 -
s -
0.9
-
a-s 5
2 0.6 CI E 0 0.3 0
1.8 -
1
*
*
1.6 1.4 3 - 1.2 E 7 1.0 ki L 0.8
Fig. 6. Effect of 17@-estradiol (1 and 10 PM, triangles and squares, respectively) on constrictor responses to BAY K 8644 (0.01, 0.03, 0.1, 0.3, and 1 PM, circles). Data shown are means & SE in g (n = 6). Experiments were performed in presence of indomethacin (10 PM). *P c 0.01 and **P < 0.05 in comparison with control group, respectively.
g 0.6 0 0.4 0.2 wi;
0 10
9.5 9 Endothelin-1
8.5 8 7.5 concentration
7 6.5 (-log M)
6
Fig. 3. Effect of verapamil (0.1, 1, and 10 PM) on concentration-dependent contractile responses to endothelin-1 in rabbit coronary arteries without endothelium. Circles, squares, triangles and diamonds indicate control, 0.1, 1, and 10 PM 17@-estradiol, respectively.
cific sites different from those linked to the dihydropyridine binding site (12,31). The calcium antagonist binding sites associated with L-type calcium channels are not the primary site of action of endothelin-1 (8). However, the extracellular calcium dependency of endothelin-1 and the dihydropyridine calcium antagonist-induced functional
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H274
17P-ESTRADIOL
ATTENUATES
ENDOTHELIN-INDUCED
inhibition of contraction strongly suggest that an increase in calcium influx is an important part of its constrictor mechanism (7, 12, 31). Verapamil, a voltage-dependent calcium channel antagonist that binds to a site allosterically linked to the dihydropyridine binding site, antagonized endothelin- l-induced contraction. In this study, we have demonstrated that verapamil shifted the endothelin- 1 concentration-dependent contraction curve to the right in a nonparallel fashion. Verapamil also suppressed maximal contraction induced by endothelin- 1. These results suggest that endothelin- 1 caused extracellular calcium influx, at least in part, by activation of voltage-dependent calcium channels in isolated rabbit coronary arterial preparations. Endothelin- 1 has been shown to increase single-channel calcium currents in coronary arterial smooth muscle via a second messenger system (22). As well as an effect on plasmalemmal calcium influx, the stimulator effect of endothelin-1 on intracellular calcium release and protein kinase C activation has been reported (1, 13). 17P-Estradiol suppresses contraction of coronary, aortic, and uterine arteries in vitro and increases uterine blood flow in vivo (9, 16, 20), although the mechanism is not well understood. Recently, it has been reported that estrogen treatment modulates endothelium-dependent relaxation in aortas from ovariectomized rabbits and in atherosclerotic coronary arteries from ovariectomized monkeys (18, 29). In the present study, however, 17pestradiol induced a concentration-dependent relaxation of endothelium-intact or -denuded rabbit coronary arteries precontracted with endothelin-1. No differences were seen between rings with and without endothelium in the presence of indomethacin. L-NMMA, a specific inhibitor of EDRF synthesis, did not inhibit UP-estradiol-induced relaxation, whereas it abolished relaxing responses to acetylcholine in rings with endothelium. Verapamil also induced a concentration-dependent and endothelium-independent relaxing effect on endothelin- 1-precontracted arteries in the preparations. These results suggest that UP-estradiol induces acute relaxation by an endotheliurn-independent mechanism in isolated rabbit coronary arteries. Our results do not rule out that UP-estradiol may modulate endothelium-dependenc vasodilation by facilitating the release of EDRF in vivo over a longer time course. In addition, 17P-estradiol stimulates prostacyclin biosynthesis in rat aortic smooth muscle cells in culture (3). However, it has also been reported that indomethacin does not affect the enhanced endothelium-dependent relaxation in rabbit aorta and femoral arteries from ovariectomized rabbits after chronic estrogen administration (6918). In the present study, similar results we re obtained in the presence or absence of indomethacin, suggesting that the acute dilator effect of 17@-estradiol is unlikely to be mediated via the release of vasodilator prostanoids. UP-Estradiol has an inhibitory effect on calcium-induced contraction of vein and uterine smooth muscle (17, 27). 17P-Estradiol has also been shown to enhance the inhibitory effect of nifedipine, a calcium entry blocker, on contraction of the rat uterine artery; the enhancement is reversed by calcium (5). In isolated, perfused heart preparations, 17@-estradiol reduces coronary perfusion pres-
CONTRACTION
sure induced by BAY K 8644, and the reduction is antagonized by calcium (21). Recently, it has been reported that UP-estradiol has an inhibitory effect on activation of voltage-sensitive calcium channels in uterine arterial smooth muscle (24, 25). In this study, 17P-estradiol shifted the calcium concentration-dependent contraction curve to the right. The EC& of calcium was increased in K+-depolarization medium. BAY K 8644, a specific agonist of voltage-dependent calcium channels (2), induced a contraction in isolated rabbit coronary arteries without endothelium. The contraction induced by BAY K 8644 was markedly reduced by 17fl-estradiol. These data suggest that UP-estradiol may suppress rabbit coronary artery contraction by affecting calcium influx. 17P-Estradiol caused a concentration-dependent attenuation of endothelin-l-evoked contraction. The concentration-dependent contractile response curves were shifted to the right in a nonparallel fashion. The -log EDS0 was increased sevenfold by UP-estradiol (10 PM). The suppression of endothelin- 1 -induced maximal contraction by 17P-estradiol suggests that the 17@-estradiol was acting in a noncompetitive manner. The antagonistic effect of UP-estradiol on endothelin-1 contraction was less than, but similar to, that of verapamil. These results suggest that the attenuation of endothelin-l-induced contraction by 17@-estradiol may be partially due to an effect on calcium influx in isolated rabbit coronary arteries. Estrogen receptors are present in cardiovascular tissues of both male and female rats (26). Administration of estrogen has been shown to induce hemodynamic changes in male human subjects after a transsexual operation (23), similar to those seen in female guinea pigs (4,10). In the present study, we have demonstrated that endothelin-1 induces identical constrictor responses and that 17/Sestradiol attenuates the contraction in coronary arteries from both male and female rabbits. Therefore, we assume that the effect of UP-estradiol on contraction induced by endothelin-1 is independent of sex in the isolated rabbit coronary artery preparation. Endothelium may regulate coronary arterial tone by the release of endothelium-derived relaxing and contracting factors. In atherosclerosis, vasospasm, and hypertension, endothelium-dependent relaxation is impaired and endothelium-dependent contraction may occur (11, 14). The plasma and myocardial concentrations of endothelin are increased in myocardial infarction, and treatment with endothelin antibodies reduces the size of myocardial infarction in rats (28). Estrogen treatment modulates endothelium-dependent vasodilation in arteries from ovariectomized rabbits (6, 18)) ovariectomized and atherosclerotic monkeys (29)) and spontaneously hypertensive rats (30). The results of this study suggest that 17@-estradiol has an acute inhibitory effect on coronary arterial contraction induced by endothelin-1. The acute inhibitory effect we have observed in isolated preparations occurs at concentrations that are higher than the plasma dose during hormone replacement therapy. The data do not prove a direct association between the in vitro acute inhibition of endothelin- 1 -induced contraction and the in vivo beneficial effect of hormone replacement therapy.
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17/3-ESTRADIOL
ATTENUATES
ENDOTHELIN-INDUCED
In conclusion, 17@-estradiol attenuates endothelin- linduced contraction in isolated rabbit coronary arteries by an endothelium-independent mechanism. Because UP-estradiol antagonizes calcium- or BAY K 8644-induced contraction in similar preparations, and verapamil, which is a calcium antagonist, induces attenuation by a same manner and time course as UP-estradiol, perhaps this acute attenuation is at least partially attributable to an inhibitory effect on calcium influx. In contrast, acetylcholine relaxes precontracted coronary arteries with endothelin-1 by releasing EDRF.
160: 1302-1308,
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l6 Magness, R. R., and C. R. Rosenfeld. Local and systemic ’ estradiol-17 beta: effects on uterine and systemic vasodilation.
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T. F., 2. Yang, M. Tschudi, L. Von-Segesser, P. Stulz, C. Boulanger, R. Siebenmann, M. Turina, and F. R. Btihler. Interaction between endothelin-1 and endothelium-derived relaxing factor in human arteries and veins. Circ. Res. 66:
Brown, A. M., D. L. Kunze, and A. Yatani. The agonist effect of dihydropyridines on Ca++ channels. Nature Lond. 311: 570-
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Address for reprint requests: P. Collins, Dept. of Cardiac Medicine, National Heart and Lung Institute, Dovehouse St., London SW3 6LY, UK. Received 20 September 1991; accepted in final form 17 February 1992.
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Res. 81: 1680-1687,
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Williams, S. P., D. P. Shackelford, S. G. Iams, and S. J. Mustafa. Endothelium-dependent relaxation in estrogen-treated spontaneously hypertensive rats. Eur. J. Pharmacol. 145: 205-207, 1988.
Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto, and T. Masaki. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature Lond. 332: 411-415, 1988. 32. Yanagisawa, M., and T. Masaki. Endothelin, a novel endothelium-derived peptide. Pharmacological activities, regulation and possible roles in cardiovascular control. Biochem. Pharmacol. 38: 1877-1883,
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JIANG,
PHILIP
M. SARREL,
PHILIP
A. POOLE-WILSON,
Department of Cardiac Medicine, National Heart and Lung Institute, London SW3 6LY, United Kingdom Jiang, Canwen, Philip M. Sarrel, Philip A. Poole-Wilson, and Peter Collins. Acute effect of 17@-estradiolon rabbit coronary artery contractile responsesto endothelin-1. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H271-H275, 1992.-We assessed the acute effect of 17@-estradiolon coronary artery constrictor responsesto endothelin-1. 17P-Estradiol significantly shifted endothelin-1, calcium, or BAY K 8644 concentration-dependent contraction curves to the right in endotheliurn-denuded coronary arteries isolated from nonpregnant female rabbits. The -log 50% effective dose(ED& of calcium in high KC1 medium (100mM) was3.8 t 0.11 in control and 3.2 & 0.1 and 2.8 t 0.12 after incubation with 17@-estradiol(1 and 10 PM, respectively). The -log EDs0 of BAY K 8644 (KC1 15 mM) was 7.8 * 0.1 in control and 7.4 t 0.08 and 7.2 t 0.05 in the presenceof 1’7@-estradiol (1 and 10 PM, respectively). The -log EDs0 of endothelin-1 was 9.2 t 0.08 in control and 8.8 t 0.1, 8.4 t 0.07, and 8.1 t 0.12 after incubation with 17@-estradiol (3, 10, and 30 PM, respectively). Similar results were obtained from coronary arteriesof malerabbits. Theseincreasesof -log EDs0 values were significant (P < 0.05 or 0.01). 17P-Estradiol and verapamil induced dose-dependentrelaxation in both endothelium-intact or -denudedcoronary arteries submaximally precontracted by endothelin-1. NG-monomethyl+arginine had no effect on relaxation induced by 17P-estradiol, whereasit eliminated relaxation induced by acetylcholine in rings with an intact endothelium. These data suggestthat 17@estradiol attenuates the rabbit coronary artery contraction induced by endothelin- 1 via an endothelium-independent mechanism, possibly by affecting calcium influx. endothelium; endothelium-derived relaxing factor; verapamil; relaxation; calcium influx ENDOTHELIUM PLAYS an important role in the regulation of coronary arterial tone by releasing both endothelium-derived relaxing and contracting factors. Endothelin-1 is a vasoconstrictor peptide isolated from the supernatant of cultured porcine endothelial cells (31, 32). It induces potent contraction in isolated coronary arteries (12). Endothelium-derived relaxing factor (EDRF) has been shown to inhibit smooth muscle contraction induced by endothelin-1 in canine arteries and veins (19) and in human arteries (15). In certain pathophysiological conditions such as atherosclerosis, endothelium-dependent vasodilation is impaired and endothelium-dependent contraction may predominate (14). Exogenous estrogen administration to guinea pigs induces similar hemodynamic changes to those seen in pregnancy (4, 10). Endothelium-dependent vasodilation is impaired in ovariectomized rabbits and spontaneously hypertensive rats, but this is partially restored by chronic estrogen administration in these animals (6, 18, 30). Intracoronary infusion of acetylcholine induces coronary artery contraction in ovariectomized and atherosclerotic monkeys but relaxation in animals treated chronically with estrogen (29). Endothelin-1 -induced contraction may occur in coronary atherosclerosis and 0363-6135/92
artery AND PETER
COLLINS
University of London,
play a role in the pathophysiology of coronary artery spasm and myocardial ischemia (11, 14, 28). However, the effect of UP-estradiol on coronary artery contraction induced by endothelin-1 is not known. In this study, we have investigated the acute effect of 17&estradiol on contractile responses to endothelin-1 in isolated coronary arterial preparations of male or nonpregnant female rabbits. MATERIALS AND METHODS Preparation of rabbit coronary arteries. Adult male or nonpregnant female New Zealand White rabbits (2.5-3 kg) were anesthetizedby an injection of pentobarbital sodium(60 mg/kg) into the ear vein after an injection of heparin (150 U/kg) into the ear vein. The hearts were removed and immediately immersedin ice-cold modified Krebs solution. The left anterior descendingcoronary artery was dissected free of connective tissue. Rings (2-3 mm length) were prepared, and in alternate rings, endothelium was removed by gently rubbing the intimal surfacewith a woodenprobe. The presenceor absenceof endothelium wasverified by the relaxation of the rings in responseto substanceP (0.3, 1, 3, and 10 nM). Measurement of arterial tension. Coronary artery rings with or without endothelium were suspendedhorizontally between two stainlesssteel hooksin individual organ baths containing 7 ml modified Krebs solution at 37’C, bubbled with 95% 02-5% C02, for measurementof isometric tension. The compositionof modified Krebs solution was (in mM) 118.3NaCl, 4.7 KCl, 2.5 CaC1,, 1.2 MgSO*, 1.2 K2P04, and 11.1 glucose.Rings were equilibrated for 90 min under a resting tension of 1 g before drugswere added.Except for one group, in which indomethacin was omitted, all experiments were performed in rings with or without endothelium in the presenceof indomethacin (10 PM). Effect of 17p-estrudiol or verapamil on endothelin-1 -induced contraction. Endothelin-1 concentration-dependentcontraction
curves in modified Krebs solution wereperformed in rings without endothelium in the absenceof l’?P-estradiol or verapamil. Becausethe tension after the contraction induced by endothelin-1 did not return to basal levels after repeatedwashout, the concentration-dependent responsecurves in the presenceand the absenceof 17P-estradiol or verapamil could not be performed in the samerings. In different tissues,after incubation with 17&estradiol (3, 10, and 30 PM; dissolved in ethanol), verapamil(O.1, 1, and 10PM; dissolvedin ethanol), or the same concentration of the solvent ethanol (1:2,000 vol) for 20 min, endothelin-1 concentration-dependent contraction curves were independently obtained. In the other group, the relaxing responsesto 17@-estradiol(1, 3, 10, 30, and 100 PM), verapamil (0.1, 0.3, 1, 3, 10, and 30 PM), or the sameconcentration of ethanol were measuredin rings after being submaximally contracted by endothelin-1 (3 nM). The relaxing responsesto 17@estradiol (1, 3, 10, 30, and 100PM) were also measuredin one group of endothelium-intact rings contracted with endothelin-1 (3 nM) in the absenceof indomethacin. iVG-monomethyl+ arginine (L-NMMA, 30 and 100 PM) was used to assessthe EDRF-dependent component of the relaxation induced by 17@estradiol or verapamil. In addition, the effects of L-arginine or D-arginine (1 mM) on the L-NMMA-induced inhibition of the
$2.00 Copyright 0 1992 the American Physiological
Society
H271
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H272
l’I@ESTRADIOL
ATTENUATES
ENDOTHELIN-INDUCED
CONTRACTION
relaxing responses to acetylcholine were measured. Effects of 17P-estradiol on contraction induced by calcium and BAY K 8644. Endothelium-denuded coronary arteries from
nonpregnant female rabbits were incubated in calcium-free solution containing 0.5 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) for 10 min. Calcium concentration-dependent contraction curves were then performed in K+-depolarization medium (KC1 100mM). The rings were equilibrated in modified Krebs solution for 20 min before being incubated in calcium-free solution containing 0.5 mM EGTA for another 10 min. These rings were incubated with 17@-estradiol(1 and 10 PM) or the sameconcentration of the solvent ethanol before the calcium concentration-dependent contraction curves. In another group of rings from female rabbits, constrictor responsesto BAY K 8644 (0.01, 0.03, 0.1, 0.3, and 1 PM plus 15 mM KCl) in the absenceor presenceof I?‘@-estradiol(1 and 10 PM) were measured. Drugs. 17@-Estradiol,verapamil, endothelin-1, acetylcholine, L-arginine, D-arginine, and indomethacin were purchasedfrom Sigma Chemical (Poole, Dorset, UK). BAY K 8644 was obtained from Calbiochem(La Jolla, CA). L-NMMA (acetatesalt) was a generousgift from Dr. S. Moncada of Wellcome Laboratories (Beckenham, Kent, UK). Statistical analyses. Values are expressedas means k SE. The relaxation is expressedaspercentageof contraction. Data wereanalyzed by the analysisof variance, followed by NewmanKeuls test. P < 0.05 was consideredto be a statistically significant difference. The number of animals is indicated by n. RESULTS
Endothelin-1 -induced contraction in rabbit coronary arteries. Endothelin- 1 induced slowly developing concen-
tration-dependent contraction in isolated rabbit coronary arterial rings. The -log 50% effective dose (ED& values were 9.3 t 0.05 and 9.3 t 0.04, respectively, in endothelium-intact or -denuded coronary arterial rings from male rabbits (n = 6; P > 0.05). In nonpregnant female preparations (n = 6), the -log EDs0 values were 9.3 t 0.07 and 9.2 + 0.08, respectively, in endothelium-intact or -denuded arterial rings (P > 0.05). Endothelin-1 (3 nM) induced comparable submaximal contraction in endothelium-intact or -denuded rings of male rabbits (1.0 t 0.2 and 1.1 t 0.1 g, respectively, P > 0.05). Effect of 1 ?P-estradiol on endothelin-1 -induced contraction. After 20-min incubation, 17P-estradiol produced
a concentration-dependent antagonism of endothelin- linduced contraction in both male and female preparations. The same concentration of the solvent ethanol had no significant effect on this contraction. The endothelin-1 -induced concentration-dependent contraction curves were shifted to the right in a nonparallel fashion. In endothelium-denuded rings of male rabbits, the -log EDs0 values of endothelin-1 were increased to 8.8 t 0.09, 8.3 t 0.11, and 8.1 t 0.07 (P < 0.05 or 0.01 in comparison with control) after incubation with 17@-estradiol (3, 10, and 30 PM, respectively). After incubation with 17@estradiol (10 or 30 p M), the maximal contractile responses to endothelin-1 were suppressed by 8 and 2l%, respectively (Fig. 1). Similar results were obtained in endothelium-denuded coronary arteries from nonpregnant female rabbits (n = 5) in which the -log EDs0 values were 8.8 t 0.1, 8.4 t 0.07, and 8.1 t 0.12 (P < 0.05 or 0.01 in comparison with control) after incubation with 17@-estradiol (3, 10, and 30 PM, respectively). UP-Estradiol in-
10
9.5 9 Endothelin-
a.5 a 7.5 1 concentration
6
Fig. 1. Effect of UP-estradiol (1, 10, and 30 PM) on concentrationdependent contractile responses to endothelin-1 in male rabbit coronary arteries without endothelium. Data shown are means t SE in g (n = 6). Experiments were performed in presence of indomethacin (10 PM). Circles, squares, triangles, and diamonds indicate control, 0.1, 1, and 10 PM 17&estradiol, respectively.
duced relaxation of endothelin- 1 (3 nM) -precontracted endothelium-intact or -denuded rings of male rabbits. No differences were observed between rings with and without endothelium (Fig. 2). Similar results were obtained in the presence or absence of indomethacin (10 PM; data not shown, n = 6). L-NMMA (30 and 100 PM) had no inhibitory effect on the 17p-estradiol-induced relaxation (data not shown, n = 6). In contrast, acetylcholine caused concentration-dependent relaxation of endothelin-l-precontracted endothelium-intact arteries. The relaxation was completely abolished by the removal of the endothelium. L-NMMA inhibited this relaxation in a concentrationdependent manner. L-arginine but not D-arginine overcame the inhibition induced by L-NMMA (data not shown, n = 6). Effect traction.
of verapamil
on endothelin-1 -induced
con-
Verapamil significantly inhibited concentration-dependent constrictor responses to endothelin-1 in a manner similar to UP-estradiol in endothelium-denuded rings of male rabbits. The concentration-dependent contraction curves were shifted to the right in a nonparallel fashion. The -log EDs0 values were increased to 8.4 t 0.03, 7.8 t 0.11, and 7.4 t 0.06 (P c 0.01 in comparison with control) after incubation with verapamil (0.1, 1, and 100 -
a0 -
20 -
Fig. 2. Relaxing effect of 17/3-estradiol on endothelin-1 (3 nM)-precontracted rabbit coronary arteries with (circles) and without (squares) endothelium. Submaximal contractions induced by endothelin-1 were 1.0 t 0.2 and 1.1 t 0.1 g in endothelium-intact and -denuded rings, respectively. Data shown are percentage relaxation means t SE (n = 6, all P > 0.05). Experiments were performed in presence of indomethacin (10 PM).
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17@-ESTRADIOL
ATTENUATES
ENDOTHELIN-INDUCED
10 PM, respectively) compared with 9.3 t 0.03 in control. However, the maximal constrictor responses to endothelin-1 were suppressed by 21 and 41% after incubation with verapamil (3 and 10 PM, respectively). This antagonistic effect of verapamil was greater than that of 17@estradiol (Fig. 3). Verapamil(O.3,1,3,10, and 30 PM) also induced relaxation of endothelin- 1-precontracted endothelium-intact or -denuded rings of male rabbits (Fig. 4). No statistically significant differences were seen between rings with and without endothelium (P > 0.05). L-NMMA (30 and 100 PM) had no inhibitory effect on verapamil-induced relaxation (data not shown, n = 6). Inhibitory effect of 17@estradiol on calcium-induced contraction. The calcium concentration-dependent con-
traction curves in K+-depolarization medium were shifted to the right after incubation with 17P-estradiol in endothelium-denuded rings of nonpregnant female rabbits. The -log 50% effective concentration (EC,,) was increased from 3.8 t 0.11 in control to 3.2 t 0.1 and 2.8 t 0.12 (P < 0.05 and 0.01, respectively) after incubation with 17P-estradiol (1 and 10 PM, respectively). Maximal contraction was reduced by 21 t 4.4 and 34 t 5.4%, respectively (Fig. 5).
H273
CONTRACTION
120 100 1
g 8o .g 60: $ 40 20 O7
6 5.5 concentration
VeradLnil
5 (-log
4.5 M)
Fig. 4. Effect of verapamil on endothelin-1 (3 nM)-precontracted rabbit coronary arteries with (triangles) and without (squares) endothelium. Submaximal contractions induced by endothelin-1 were 1.1 t 0.2 and 1.1 k 0.2 g in endothelium-intact and -denuded rings, respectively. Data shown are percentage relaxation means t SE (n = 6, P > 0.05).
Inhibitory effect of 17@-estradiol on BAY K 8644-induced contraction. BAY K 8644 (0.01, 0.03, and 0.1 PM
plus 15 mM KCl) induced concentration-dependent contraction in endothelium-denuded rings of nonpregnant female rabbits. Higher concentrations of BAY K 8644 (0.3 and 1 PM) did not cause further contraction and induced slight reduction of contraction evoked by lower concentrations in some preparations. In the presence of 17@-estradiol (1 and 10 PM), BAY K 8644-induced contraction was markedly reduced (P < 0.05 or 0.01, respectively, Fig. 6). The -log ECSO values were 7.8 t 0.1 in control and 7.4 t 0.08 and 7.2 t 0.05 in the presence of 17P-estradiol (1 and 10 PM, respectively).
0 ’ 4
3.5 Calcium
3 2.5 Concentrations
2 (-log
1.5 M)
Fig. 5. Effect of 17P-estradiol (1 and 10 PM, squares and triangles, respectively) on calcium concentration-dependent contraction (control, circles) in high KC1 (100 mM) medium, in endothelium-denuded coronary arteries of female rabbits. Data are expressed as percentage contraction (means k SE, n = 5) of maximal contraction induced by calcium in control condition. Experiments were performed in presence of indomethacin (10 PM).
DISCUSSION
We have demonstrated that endothelin-1 is a potent constrictor of the rabbit coronary artery. Our results are consistent with a previous report in the porcine coronary artery (12). Activation of voltage-dependent calcium channels is thought to be a part of the mechanism of endothelin-1 contraction. This involves binding to spe2.0 -
s -
0.9
-
a-s 5
2 0.6 CI E 0 0.3 0
1.8 -
1
*
*
1.6 1.4 3 - 1.2 E 7 1.0 ki L 0.8
Fig. 6. Effect of 17@-estradiol (1 and 10 PM, triangles and squares, respectively) on constrictor responses to BAY K 8644 (0.01, 0.03, 0.1, 0.3, and 1 PM, circles). Data shown are means & SE in g (n = 6). Experiments were performed in presence of indomethacin (10 PM). *P c 0.01 and **P < 0.05 in comparison with control group, respectively.
g 0.6 0 0.4 0.2 wi;
0 10
9.5 9 Endothelin-1
8.5 8 7.5 concentration
7 6.5 (-log M)
6
Fig. 3. Effect of verapamil (0.1, 1, and 10 PM) on concentration-dependent contractile responses to endothelin-1 in rabbit coronary arteries without endothelium. Circles, squares, triangles and diamonds indicate control, 0.1, 1, and 10 PM 17@-estradiol, respectively.
cific sites different from those linked to the dihydropyridine binding site (12,31). The calcium antagonist binding sites associated with L-type calcium channels are not the primary site of action of endothelin-1 (8). However, the extracellular calcium dependency of endothelin-1 and the dihydropyridine calcium antagonist-induced functional
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H274
17P-ESTRADIOL
ATTENUATES
ENDOTHELIN-INDUCED
inhibition of contraction strongly suggest that an increase in calcium influx is an important part of its constrictor mechanism (7, 12, 31). Verapamil, a voltage-dependent calcium channel antagonist that binds to a site allosterically linked to the dihydropyridine binding site, antagonized endothelin- l-induced contraction. In this study, we have demonstrated that verapamil shifted the endothelin- 1 concentration-dependent contraction curve to the right in a nonparallel fashion. Verapamil also suppressed maximal contraction induced by endothelin- 1. These results suggest that endothelin- 1 caused extracellular calcium influx, at least in part, by activation of voltage-dependent calcium channels in isolated rabbit coronary arterial preparations. Endothelin- 1 has been shown to increase single-channel calcium currents in coronary arterial smooth muscle via a second messenger system (22). As well as an effect on plasmalemmal calcium influx, the stimulator effect of endothelin-1 on intracellular calcium release and protein kinase C activation has been reported (1, 13). 17P-Estradiol suppresses contraction of coronary, aortic, and uterine arteries in vitro and increases uterine blood flow in vivo (9, 16, 20), although the mechanism is not well understood. Recently, it has been reported that estrogen treatment modulates endothelium-dependent relaxation in aortas from ovariectomized rabbits and in atherosclerotic coronary arteries from ovariectomized monkeys (18, 29). In the present study, however, 17pestradiol induced a concentration-dependent relaxation of endothelium-intact or -denuded rabbit coronary arteries precontracted with endothelin-1. No differences were seen between rings with and without endothelium in the presence of indomethacin. L-NMMA, a specific inhibitor of EDRF synthesis, did not inhibit UP-estradiol-induced relaxation, whereas it abolished relaxing responses to acetylcholine in rings with endothelium. Verapamil also induced a concentration-dependent and endothelium-independent relaxing effect on endothelin- 1-precontracted arteries in the preparations. These results suggest that UP-estradiol induces acute relaxation by an endotheliurn-independent mechanism in isolated rabbit coronary arteries. Our results do not rule out that UP-estradiol may modulate endothelium-dependenc vasodilation by facilitating the release of EDRF in vivo over a longer time course. In addition, 17P-estradiol stimulates prostacyclin biosynthesis in rat aortic smooth muscle cells in culture (3). However, it has also been reported that indomethacin does not affect the enhanced endothelium-dependent relaxation in rabbit aorta and femoral arteries from ovariectomized rabbits after chronic estrogen administration (6918). In the present study, similar results we re obtained in the presence or absence of indomethacin, suggesting that the acute dilator effect of 17@-estradiol is unlikely to be mediated via the release of vasodilator prostanoids. UP-Estradiol has an inhibitory effect on calcium-induced contraction of vein and uterine smooth muscle (17, 27). 17P-Estradiol has also been shown to enhance the inhibitory effect of nifedipine, a calcium entry blocker, on contraction of the rat uterine artery; the enhancement is reversed by calcium (5). In isolated, perfused heart preparations, 17@-estradiol reduces coronary perfusion pres-
CONTRACTION
sure induced by BAY K 8644, and the reduction is antagonized by calcium (21). Recently, it has been reported that UP-estradiol has an inhibitory effect on activation of voltage-sensitive calcium channels in uterine arterial smooth muscle (24, 25). In this study, 17P-estradiol shifted the calcium concentration-dependent contraction curve to the right. The EC& of calcium was increased in K+-depolarization medium. BAY K 8644, a specific agonist of voltage-dependent calcium channels (2), induced a contraction in isolated rabbit coronary arteries without endothelium. The contraction induced by BAY K 8644 was markedly reduced by 17fl-estradiol. These data suggest that UP-estradiol may suppress rabbit coronary artery contraction by affecting calcium influx. 17P-Estradiol caused a concentration-dependent attenuation of endothelin-l-evoked contraction. The concentration-dependent contractile response curves were shifted to the right in a nonparallel fashion. The -log EDS0 was increased sevenfold by UP-estradiol (10 PM). The suppression of endothelin- 1 -induced maximal contraction by 17P-estradiol suggests that the 17@-estradiol was acting in a noncompetitive manner. The antagonistic effect of UP-estradiol on endothelin-1 contraction was less than, but similar to, that of verapamil. These results suggest that the attenuation of endothelin-l-induced contraction by 17@-estradiol may be partially due to an effect on calcium influx in isolated rabbit coronary arteries. Estrogen receptors are present in cardiovascular tissues of both male and female rats (26). Administration of estrogen has been shown to induce hemodynamic changes in male human subjects after a transsexual operation (23), similar to those seen in female guinea pigs (4,10). In the present study, we have demonstrated that endothelin-1 induces identical constrictor responses and that 17/Sestradiol attenuates the contraction in coronary arteries from both male and female rabbits. Therefore, we assume that the effect of UP-estradiol on contraction induced by endothelin-1 is independent of sex in the isolated rabbit coronary artery preparation. Endothelium may regulate coronary arterial tone by the release of endothelium-derived relaxing and contracting factors. In atherosclerosis, vasospasm, and hypertension, endothelium-dependent relaxation is impaired and endothelium-dependent contraction may occur (11, 14). The plasma and myocardial concentrations of endothelin are increased in myocardial infarction, and treatment with endothelin antibodies reduces the size of myocardial infarction in rats (28). Estrogen treatment modulates endothelium-dependent vasodilation in arteries from ovariectomized rabbits (6, 18)) ovariectomized and atherosclerotic monkeys (29)) and spontaneously hypertensive rats (30). The results of this study suggest that 17@-estradiol has an acute inhibitory effect on coronary arterial contraction induced by endothelin-1. The acute inhibitory effect we have observed in isolated preparations occurs at concentrations that are higher than the plasma dose during hormone replacement therapy. The data do not prove a direct association between the in vitro acute inhibition of endothelin- 1 -induced contraction and the in vivo beneficial effect of hormone replacement therapy.
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17/3-ESTRADIOL
ATTENUATES
ENDOTHELIN-INDUCED
In conclusion, 17@-estradiol attenuates endothelin- linduced contraction in isolated rabbit coronary arteries by an endothelium-independent mechanism. Because UP-estradiol antagonizes calcium- or BAY K 8644-induced contraction in similar preparations, and verapamil, which is a calcium antagonist, induces attenuation by a same manner and time course as UP-estradiol, perhaps this acute attenuation is at least partially attributable to an inhibitory effect on calcium influx. In contrast, acetylcholine relaxes precontracted coronary arteries with endothelin-1 by releasing EDRF.
160: 1302-1308,
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