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Cardiovascular Research 1999 42(1):224-231; doi:10.1016/S0008-6363(98)00265-X
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Copyright © 1999, European Society of Cardiology

Short-term exposure to physiological levels of 17β-estradiol enhances endothelium-independent relaxation in porcine coronary artery1

Hwee Teoh*, Susan W.S. Leung and Ricky Y.K. Man

Department of Pharmacology, Faculty of Medicine, The University of Hong Kong, 1/F Li Shu Fan Building, 5 Sassoon Road, Hong Kong, PR China

* Corresponding author. Tel.: +852-2819-9103; fax: +852-2817-0859. E-mail address: hteoh@hkusua.hku.hk (H. Teoh)

Received 10 June 1998; accepted 6 August 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: While alterations in cholesterol and lipoprotein profiles partly account for menopause being a risk factor for coronary heart disease, recent studies have suggested that 17β-estradiol may have vascular effects. Our aims were to study the short-term effects of 17β-estradiol on vascular function in isolated porcine coronary artery rings. Concomitantly, we sought to determine if physiological concentrations of 17β-estradiol could acutely potentiate relaxation. Results: 17{alpha}- and 17β-estradiol at pharmacological (>1 µM) concentrations produced relaxation in U46619 [GenBank] -pre-contracted porcine coronary artery rings. Relaxation evoked by 17β-estradiol was not reversed by the estrogen receptor antagonists tamoxifen and ICI 182780. Following 20 min exposure to a physiological concentration of 17β-estradiol (1 nM), which on its own had no effect, relaxation elicited by cromakalim, levcromakalim and sodium nitroprusside, but not bradykinin or calcium ionophore A23187 [GenBank] , were significantly enhanced. This potentiating action was also insensitive to tamoxifen and ICI 182780. Our data provide evidence for an acute indirect relaxant action of 17β-estradiol and suggest that it may be via a tamoxifen- and ICI 182780-insensitive estrogen receptor. While this response was only observed at pharmacological concentrations, the potentiation of cromakalim, levcromakalim and sodium nitroprusside relaxation was evident in the presence of a physiological concentration (1 nM) of 17β-estradiol. Conclusions: These results demonstrate that short-term exposure to 17β-estradiol, at concentrations that have no effect on their own, can enhance vasorelaxation. These vascular effects may partly account for some of the acute effects of 17β-estradiol on blood flow.

KEYWORDS Estradiol; Vessel; Coronary; Vasodilation; Swine


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Prior to menopause, women have a lower incidence of coronary heart disease (CHD) compared to age-matched men [1–3]. Estrogen replacement therapy reduces CHD mortality in post-menopausal women [4, 5]and diminishes coronary artery atherosclerosis in primates [6]. The protective effects of estrogen are partially attributable to its favorable alterations of serum lipoprotein levels [4, 7, 8], its anti-oxidant properties [9, 10]and its actions as a calcium antagonist [11]on voltage operated calcium channels [12, 13].

Increasingly, however, many investigators have studied the vascular effects of 17β-estradiol. Chronic [14–16]and acute [17–19]administration of 17β-estradiol at physiological levels in vivo enhances endothelium-dependent vasodilation. Conversely, supraphysiological concentrations of the steroid probably act via the smooth muscle cells to elicit relaxation [11, 12, 20]. Hence, it has been proposed that the vascular actions of estrogen may contribute, in part, to the cardioprotective effects in pre-menopausal women [21].

Currently, the mechanism(s) of the acute and chronic vascular effects of estrogen at physiological or conventional pharmacological plasma concentrations remain unclear. The aim of the present study was to determine if physiological concentrations of 17β-estradiol could acutely potentiate relaxation in vitro. The rationale for using pigs as an animal model was based on the similarities between the porcine and human hearts.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
U46619 (9,11-dideoxy-9{alpha},11{alpha}-methanoepoxy prostaglandin F2{alpha}) was obtained from Biomol (PA, USA) and ICI 182780 was a gift from Zeneca (Macclesfield, UK). Cromakalim and levcromakalim were generous gifts from SmithKline Beecham, UK. All other chemicals were from Sigma (St. Louis, MO, USA). Stocks of 17{alpha}-estradiol, 17β-estradiol, ICI 182780, cromakalim, levcromakalim and U46619 [GenBank] were made up in absolute ethanol while tamoxifen was dissolved in 10% ethanol, A23187 [GenBank] in DMSO and indomethacin in a buffered sodium carbonate solution. All working solutions were obtained by dilutions in Krebs–Henseleit buffer.

2.2 Functional studies
Pigs were processed according to the regulations laid down by the Department of Agriculture, Government of Hong Kong. Porcine hearts of either sex were collected from the local abattoir and rinsed in cold, oxygenated (O2:CO2, 95%:5%) Krebs–Henseleit solution (KHS; composition in mM: 120 NaCl, 4.76 KCl, 25 NaHCO3, 1.18 NaH2PO4·H2O, 1.25 CaCl2, 1.18 MgSO4·H2O, 5.5 glucose) before the left anterior descending and right coronary arteries were isolated. After removal of connective tissue, intact coronary artery rings (3-mm wide) were suspended between stainless steel stirrups and stationary support rods positioned in 5-ml jacketed organ baths filled with oxygenated KHS maintained at 37°C. To investigate drug effects on endothelium-independent relaxation, some porcine coronary arteries were divided into two sections and perfused for 30 s at 0.5 ml/s with either 0.5% Triton X-100 in KHS or KHS alone. Artery rings were placed under a 2 g tension for 120 min before commencement of the experiment. Bath KHS was changed every 15 min during the whole equilibration period. Isometric tension was measured by force transducers (FT03, Grass, USA) coupled to an amplifier and a personal computer for data collection (PICO Data Logger, Pico Technology, Cambridge, UK).

The viability of each porcine coronary artery ring was determined by pre-contracting with 30 nM U46619 [GenBank] in the presence of 10 µM indomethacin (a cyclooxygenase inhibitor) and relaxing with 1 µM bradykinin. Only rings that produced ≥4 g contraction and demonstrated ≥80% relaxation were used for further studies. Porcine coronary artery rings were considered to have damaged endothelial layers after perfusion with Triton X-100 if they exhibited less than 20% relaxation in response to the same concentration of bradykinin. After removal of these drugs by repeated washings, the rings were contracted under the same conditions as described above and the relaxational properties were measured by cumulative additions of the appropriate agents. In some experiments, 17β-estradiol (final concentration of 0.1–100 nM) was added to the baths 20 min prior to contraction with U46619 [GenBank] and remained present throughout the experiment. Whenever the experimental protocol required the use of antagonists, these agents were added together with indomethacin 20 min before the addition of estradiol.

2.3 Data and statistical analyses
All data shown denote mean±S.E.M. with n indicating the number of porcine hearts. Relaxations elicited by the various vasodilators were expressed as a percentage of U46619 [GenBank] -induced contraction. EC50 values were determined using a curve-fitting program (SIGMAPLOT, Jandel Scientific Software, CA, USA). The results were analyzed with Student’s t-test for paired and unpaired observations. Analysis of variance (ANOVA) and Dunnett’s test were applied where appropriate to determine individual differences between multiple groups of data using a computer statistical package (SIGMASTAT, Jandel Scientific Software). A P value of <0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of estradiol isomers on pre-contracted artery rings
U46619 (30 nM) produced 6.00±0.05 g contractions (n=75) in porcine coronary artery rings. As illustrated in Fig. 1, 17β-estradiol (0.001–100 µM) produced full relaxation of U46619 [GenBank] -pre-contracted rings in a concentration-dependent manner while 100 µM 17{alpha}-estradiol only elicited relaxations of 29.31±2.55% (n=8). Additions of the estradiol vehicle instead of estradiol showed that it did not influence the effects of either stereoisomer significantly (data not shown). The action of 17β-estradiol was not dependent on the presence of intact endothelium since pretreatment with Triton X-100 had no significant effect (data not shown). This direct relaxant effect of 17β-estradiol in porcine coronary artery rings was both tamoxifen- and ICI 182780-insensitive (Fig. 2).


Figure 1
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  		Fig. 1 Direct relaxing effects of 17{alpha}-estradiol ({circ}) and 17β-estradiol (bullet) on U46619 (30 nM) pre-contracted porcine coronary artery rings. Mean U46619-generated force in the rings treated with 17{alpha}- estradiol and 17β-estradiol were 6.04±0.23 g and 5.92±0.19 g, respectively (n=8). *P<0.05 vs. corresponding 17β-estradiol effect.

 

Figure 2
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  		Fig. 2 Effects of 17β-estradiol in the absence ({blacktriangleup}) and presence of tamoxifen ({blacksquare}, 1 nM; {square}, 10 µM) or ICI 182780 (bullet, 1 nM; {circ}, 10 µM) on U46619 (30 nM) pre-contracted porcine coronary artery rings (n=7).

 
3.2 Effect of physiological concentrations of 17β-estradiol on porcine coronary artery rings
Bradykinin (0.1 nM–1 µM), A23187 [GenBank] (0.1 nM–1 µM), cromakalim (0.1 nM–100 µM), levcromakalim (0.1 nM–100 µM) and sodium nitroprusside (1 nM–100 µM) all relaxed U46619 [GenBank] pre-contracted tissues (Figs. 3–5GoGo). Pre-incubation with 17β-estradiol (1–100 nM) for 20 min did not affect U46619 [GenBank] contraction. The relaxation effects elicited by bradykinin and A23187 [GenBank] were also not significantly altered by 17β-estradiol (Fig. 3). However, 1 nM of 17β-estradiol significantly potentiated cromakalim-, levcromakalim- and sodium nitroprusside-induced endothelium-independent relaxations (Figs. 4 and 5Go). Incubation of porcine coronary artery rings with this concentration of 17β-estradiol for 20 min reduced the EC50 values of cromakalim, levcromakalim and sodium nitroprusside from 0.71±0.05, 0.56±0.05 and 1.86±0.10 µM to 0.34±0.03, 0.32±0.02 and 0.60±0.08 µM, respectively (n=6, 6 and 8, respectively; P<0.05 in all cases). Interestingly, the leftward shifts of the cromakalim, levcromakalim and sodium nitroprusside concentration–response curves following exposure to 1 nM 17β-estradiol were also apparent in the presence of 10 µM tamoxifen or 10 µM ICI 182780 (Fig. 6). As shown in Fig. 5, 0.1 nM 17β-estradiol did not significantly affect sodium nitroprusside relaxation. On the other hand, the enhancement observed with 1 nM 17β-estradiol was similar to that induced by 10 nM of the steroid, an effect which was also present in ‘endothelium-free’ porcine coronary artery rings (Fig. 7). In contrast to 1 nM 17β-estradiol, the same concentration of 17{alpha}-estradiol failed to significantly influence the relaxation elicited by sodium nitroprusside (Fig. 8). In this series of experiments, the EC50 value under control conditions was 1.51±0.02 µM while those after treatment with 17β-estradiol and 17{alpha}-estradiol were 0.67±0.06 µM (P<0.05 when compared with control data) and 1.41±0.01 µM (P>0.05 when compared with control data) respectively.


Figure 3
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  		Fig. 3 Effects of 17β-estradiol on endothelium-dependent relaxation of porcine coronary artery rings. Tissues were incubated with 17β-estradiol ({blacksquare}, 1 nM; {blacktriangleup}, 10 nM; {diamondsuit}, 100 nM) or an equal volume of ethanol solvent ({circ}) for 20 min before contracting with 30 nM U46619. Rings were then relaxed with (a) bradykinin or (b) A23187. (n=8 for each point).

 

Figure 4
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  		Fig. 4 Effects of 17β-estradiol on endothelium-independent relaxation of porcine coronary artery rings. Tissues were incubated with 1 nM 17β-estradiol (bullet) or an equal volume of ethanol solvent ({circ}) for 20 min before contracting with 30 nM U46619. Rings were then relaxed with (a) cromakalim (n=6) or (b) levcromakalim (n=6). *P<0.05 vs. corresponding control data.

 

Figure 5
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  		Fig. 5 Effects of 17β-estradiol on sodium nitroprusside-induced relaxation of porcine coronary artery rings. Tissues were incubated with 17β-estradiol (bullet, 0.1 nM; {blacksquare}, 1 nM; {blacktriangleup}, 10 nM) or an equal volume of ethanol solvent ({circ}) for 20 min before contracting with 30 nM U46619. Rings were then relaxed with sodium nitroprusside (n=8). *P<0.05 vs. corresponding control data.

 

Figure 6
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  		Fig. 6 Effects of 1 nM 17β-estradiol in the absence and presence of 10 µM tamoxifen or 10 µM ICI 182780 on endothelium-independent relaxation of porcine coronary artery rings. Tissues were incubated with 17β-estradiol (bullet), 17β-estradiol and tamoxifen ({blacktriangleup}), 17β-estradiol and ICI 182780 ({diamondsuit}) or ethanol solvent ({circ}) for 20 min before contracting with 30 nM U46619. Rings were then relaxed with (a) cromakalim (n=7), (b) levcromakalim (n=8) or (c) or sodium nitroprusside (n=9).

 

Figure 7
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  		Fig. 7 Effects of 17β-estradiol on sodium nitroprusside-induced relaxation of intact ({circ}, bullet) and denuded ({square}, {blacksquare}) porcine coronary artery rings. Tissues were incubated with 1 nM 17β-estradiol (bullet, {blacksquare}) or an equal volume of ethanol solvent ({circ}, {square}) for 20 min before contracting with 30 nM U46619. Rings were then relaxed with sodium nitroprusside in a cumulative manner (n=8). Mean U46619-generated force in intact and denuded rings were 5.97±0.14 g and 6.62±0.16 g, respectively. {dagger}, *P<0.05 vs. corresponding control data from intact and denuded rings, respectively.

 

Figure 8
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  		Fig. 8 Stereospecific effect of estradiol on sodium nitroprusside-induced relaxation of porcine coronary artery rings. Tissues were incubated with estradiol vehicle ({blacktriangleup}), 1 nM 17{alpha}-estradiol ({circ}) or 1 nM 17β-estradiol (bullet) for 20 min before contracting with 30 nM U46619. Rings were then relaxed with sodium nitroprusside in a cumulative manner (n=5). *P<0.05 vs. control.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We report here that acute treatment of porcine coronary artery rings with a low concentration (1 nM) of 17β-estradiol can affect vascular function indirectly through enhancement of vasorelaxation to cromakalim, levcromakalim and sodium nitroprusside. In accord with previous findings, we also demonstrate that estradiol can induce direct relaxation in a stereospecific and concentration-dependent manner. In addition, both the direct and indirect vascular effects of 17β-estradiol appear to be mediated via a tamoxifen- and ICI 182780-insensitive mechanism. More importantly, while the direct effects of this sex hormone only occurred at pharmacological concentrations, the indirect enhancing effects were apparent at a concentration within the physiological range.

Our data showing that 17β-estradiol, but not 17{alpha}-estradiol, can significantly produce direct relaxation (Fig. 1) is consistent with those previously reported for agonist-stimulated and high extracellular K+-depolarized rat aorta rings and rabbit coronary artery rings [20, 22]. The exception to this stereospecificity is the study by Salas et al. [23]that demonstrated both isomers were equally potent. Other than possible differences in experimental conditions, the explanation for the discrepancy in this study is not apparent.

Estrogen receptors have been detected in human coronary artery and umbilical vein endothelial cell cultures [24]as well as smooth muscle cells of rat aortae [25, 26], human coronary arteries [27], saphenous veins and mammary arteries [28, 29]. However, immunohistochemical evidence suggest that the classical estrogen receptor is absent in human coronary arteries and that the steroid-induced relaxation is independent of this [30]. Indeed, the rapid effects observed upon acute application of 17β-estradiol are inconsistent with those mediated by the intrinsically slow gene induction process in classic target tissues of 17β-estradiol [12, 17, 18, 31]. While 17β-estradiol-evoked calcium ion mobilization in porcine coronary arteries was insensitive to tamoxifen [12], another estrogen receptor antagonist ICI 182,780 decreased the proliferative actions of the sex hormone in human endothelial cell cultures [26]. Since estrogen receptors appear to be heterogenous [32–34]and the smooth muscle relaxant effect of 17β-estradiol are probably not due to non-specific steroidal effects [22], these acute responses do not appear to involve the classical estrogen receptor.

Bradykinin is believed to cause relaxation via the release of both NO and endothelium-dependent hyperpolarizing factor [35]while A23187 [GenBank] stimulates NO synthesis by raising intracellular calcium concentrations. The absence of an estrogen effect on the endothelium-dependent relaxations induced by both bradykinin and A23187 [GenBank] (Fig. 3) suggests that the augmentations elicited by 17β-estradiol were not due to increased NO release or activity. This is in accord with our observations with the potassium channel openers, cromakalim and levcromakalim [36]. Interestingly, while pregnancy results in an increase in NO synthase activity in the guinea-pig uterine artery [37], acetylcholine-mediated relaxation in the same tissue has been found to remain unchanged [38]. Hence, it has been suggested that pregnancy may induce NO synthase activity in uterine vascular smooth muscle and as such not involve NO activity within the endothelium [39].

Previous studies found that A23187 [GenBank] -stimulated relaxation in rabbit femoral and canine coronary artery is not altered by long-term treatment with 17β-estradiol [40, 41]. In contrast, Bell and colleagues [42]have recently reported that only porcine coronary artery rings exposed to 1 nM 17β-estradiol for 18–22 h exhibited enhanced response to A23187. [GenBank] Hence, to the best of our knowledge, this is the first report demonstrating that physiological concentrations of 17β-estradiol can acutely (<30 min) modulate relaxation induced by vasodilating agents in vitro.

Administration of exogenous 17β-estradiol to achieve physiological concentrations rapidly (<30 min) improved acetylcholine-induced-endothelium-dependent relaxation in female primates [19]and humans [17, 18, 43]. In contrast, Bell et al. [42]reported that overnight, but not acute, exposure to 1 nM 17β-estradiol enhanced A23187 [GenBank] -mediated endothelium-dependent relaxation in porcine coronary artery ring segments. Similarly, we found that endothelium-dependent relaxation produced by A23187 [GenBank] was not affected by short-term treatment with 1 nM 17β-estradiol (Fig. 3). However, we observed a significant alteration in the concentration–response curves and EC50 values elicited by cromakalim, levcromakalim and sodium nitroprusside, all of which are endothelium-independent relaxing agents. A common feature of our work and that of Bell et al. [42]is the absence of enhancement of endothelium-dependent relaxation following acute incubation with a physiological concentration of 17β-estradiol. These findings contradict those obtained from clinical studies [17–19, 43]and may in part be attributed to the in vitro systems used by Bell’s group and ourselves. A point worthwhile noting is that acetylcholine was the only agent used in the above mentioned in vivo studies to determine the extent of endothelium-dependent vasorelaxation. As such, the positive modulatory activities of 17β-estradiol on endothelium-dependent relaxation reported previously may be peculiar to acetylcholine. We were, however, unable to try and mimic the in vivo situation since acetylcholine causes contraction and not relaxation in porcine coronary arteries.

High concentrations of 17β-estradiol are needed to produce direct vasorelaxation actions (Fig. 1). In contrast, short-term (20 min) incubation with 1 nM of the active estradiol isomer significantly enhanced vasorelaxation to cromakalim, levcromakalim and sodium nitroprusside (Figs. 4–8GoGoGoGo). This concentration of 17β-estradiol (1 nM) compares well with the physiological ranges in castrated and ovariectomized pigs ({approx}0.2 nM) [44]as well as that in pregnant pigs ({approx}2–10 nM) [45]. The potentiation of relaxation appeared to be of a sharp concentration–response nature since 0.1 nM 17β-estradiol had no significant effect on sodium nitroprusside relaxation while those in the presence of 1 nM and 10 nM of 17β-estradiol were enhanced to a similar level (Fig. 5). Interestingly, bradykinin- and A23187 [GenBank] -induced relaxation were unaffected by 1–100 nM of 17β-estradiol (Fig. 3). We did not attempt to examine the effects of higher concentrations of 17β-estradiol since these would not only directly cause relaxations but would also be physiologically irrelevant. As the porcine hearts were from a local abattoir, we were unable to determine or control the sex distribution of the hearts we obtained. While we recognize this as a limitation of our study, the data we present herein were reproducible in every batch of hearts we studied.

In conclusion, the acute enhancing effects of such low concentrations of 17β-estradiol (1 and 10 nM) in an in vitro preparation were in itself intriguing especially since these concentrations of 17β-estradiol did not, on their own, influence the contractile response. Unfortunately, we are unable at present, to provide a critical explanation as to why these potentiating effects were only evident when agents eliciting endothelium-independent relaxation were used. Since both the direct and indirect effects of 17β-estradiol had relatively rapid onsets and were insensitive to tamoxifen and ICI 182780, it would appear that they might be mediated by an estrogen receptor that acts via a non-genomic mechanism. However, because the direct action of 17β-estradiol occurred at supraphysiological concentrations and the indirect effect was evident at physiological levels, it remains to be determined if these two responses are mediated via the same receptor. The current knowledge of possible vascular estrogen receptor subtypes, their expression, cellular location(s) and post-receptor activities is still in the early phase and the existence of a fast-acting surface membrane estrogen receptor is still speculative. Nevertheless, the enhancing vasorelaxant action of 17β-estradiol observed in our model may partly account for some of the acute beneficial effects of 17β-estradiol on blood flow.

Time for primary review 28 days.


   Acknowledgements
 
The authors would like to thank Godfrey S.K. Man for excellent technical assistance and Adrian Quan for aiding in the graphics. This study was supported by a Committee on Research and Conference Grant, University of Hong Kong. HT and SWSL are recipients of a post-doctoral fellowship award and a postgraduate studentship, respectively, from the University of Hong Kong. RYKM is a member of the Institute of Cardiovascular Science and Medicine, University of Hong Kong.


   Notes
 
1 See pages 9–11. Back


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

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