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CARDIOVASCULAR
Agonists and Is Abolished by Estrogen Deprivation
Department of Pharmacological Sciences, University of Milan, Milan, Italy (C.B., A.C., P.S., V.P., F.P., L.P., C.P.); and Department of Pharmacology and Anesthesiology, University of Padua, Padua, Italy (A.C.)
Received December 24, 2004; accepted February 15, 2005.
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Abstract |
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agonist 4,4',4''-(4-propyl-[1H]pyrazole-1,3,5-triyl) tris-phenol (PPT) to precontracted aortic rings from intact female rats dose-dependently induced an ER-dependent vascular relaxation fully overlapping to that induced by 17
-estradiol. By contrast, the selective ER agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) had no acute effect on vasomotion. This short-term vasorelaxant action of PPT was abolished by the NO synthase inhibitor N
-nitro-L-arginine methyl ester and by endothelium removal. In aortic tissues from ovariectomized (OVX) rats, however, neither 17-estradiol nor PPT induced acute vascular relaxation. The effect of PPT was restored in preparations from estrogen-replaced OVX rats, whereas DPN remained ineffective even after estrogen replacement. PPT acted through an ER-dependent mechanism, as shown by impaired response in the presence of the anti-estrogen ICI 182,780 (7,17-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol). Accordingly, isolated rat aortic endothelial cells expressed both ER and ER. These data show that selective ER but not ER agonists reproduced the acute vasodilation of estrogen via a receptor-mediated pathway in the aorta from intact as well as 17-estradiol-replaced OVX rats. This beneficial effect was undetectable in tissues from OVX rats. Selective pharmacological targeting of ER subtypes may thus represent a novel and promising approach in the treatment of vascular disease.
; Cignarella et al., 2001). Although different mechanisms have been reported (Shaw et al., 2001), such effects seem to be mediated by specific estrogen receptors (ERs) that are located on the plasma membrane as well as intracellularly. So far, two ER subtypes have been described: ER and ER. Both subtypes are found in vascular smooth muscle (Register and Adams, 1998; Hodges et al., 2000; Maggi et al., 2003) and human endothelial cells (Caulin-Glaser et al., 1996). Although similar, the two ER isoforms are distinct gene products with nonoverlapping functions. They are coexpressed in most tissues but increasing evidence suggests that ER and ER mediate opposite effects in a kind of yin-yang manner (Gustafsson, 2003).
The relative contribution of each ER subtype to vascular responses has been difficult to investigate because the physiological ER ligand 17-estradiol (E2) has no binding affinity preference for ER and ER. Selective ER agonists have now become available, the most widely used being 4,4',4''-(4-propyl-[1H]pyrazole-1,3,5-triyl) tris-phenol (PPT), a relatively selective ER agonist, and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), a relatively selective ER agonist. These compounds have been shown to acutely relax precontracted isolated mesenteric arteries from male rats (Montgomery et al., 2003). Such effects, however, were raised at pharmacological (i.e., micromolar) concentrations, thereby questioning their possible relevance to the biology of the vascular wall. Furthermore, the hormonal status has been previously shown to influence the responsiveness of isolated rat mesenteric arteries to E2 (Shaw et al., 2001). Since E2 modulates ER expression in vascular and nonvascular tissues, ER-dependent responses may be closely related to the hormonal status (Ihionkhan et al., 2002).
To unravel the pharmacological actions of compounds selective for individual ER isotypes, and taking into account the influence of hormonal status on estrogen-mediated vasomotor responses, we set out to investigate the acute effects of selective ER agonists in a wide concentration range on vascular tone in the aorta of intact and ovariectomized (OVX) female rats.
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Materials and Methods |
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-nitro-L-arginine methyl ester (L-NAME), and E2 were purchased from Sigma Chemical (Milan, Italy). PPT, DPN, and ICI 182,780 were purchased from Tocris Cookson Inc. (Bristol, UK). NA, acetylcholine, and L-NAME were dissolved in distilled H2O, whereas E2 was freshly dissolved in ethanol to a concentration of 10–3 M. PPT, DPN, and ICI 182,780 were also dissolved in ethanol. The final ethanol concentration in organ baths did not exceed 0.01%.
Cell Culture. Rat aortic endothelial cells (RAECs) were obtained essentially as described by Ding and Vaziri (1998), with minor modifications. The cells were used for the experiments once they had formed a monolayer.
Animals. Experiments were performed on isolated aortic rings excised from female Sprague-Dawley rats weighing 200 to 250 g (Charles River Italia, Calco, Italy). The animals were kept in temperature-controlled facilities on a 12-h light/dark cycle and fed normal chow. Bilateral ovariectomy was performed under ketamine (40 mg/kg i.p.) and xylazine (20 mg/kg i.p.) anesthesia. Four weeks later, the castrated animals received a subcutaneous implant of two silastic capsules containing 25 µl of vehicle (peanut oil) or E2 (5.87 µg; 0.86 mM) for 5 days (Bolego et al., 1999; Cignarella et al., 2000). Plasma E2 concentrations after this treatment approached the normal rat proestrus level (Lapchak, 1991). All procedures were performed in accordance with the guidelines for laboratory animal care of Milan University.
Isolated Organ Bath Experiments. The aorta and the superior mesenteric artery were carefully removed, cleaned of fat and connective tissue, and cut into 5- to 6-mm rings. Vessels were suspended in 5-ml organ baths containing Krebs' solution at 37°C, continuously bubbled with 95% O2 and 5% CO2. The Krebs' solution had the following composition: 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.1 mM MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3, and 5.5 mM glucose, pH 7.4. The rings were connected to isometric tension transducers (Fort 10; WPI, Sarasota, FL) coupled with a digital recording system (PowerLab 8SP; ADInstruments, Basile, Comerio, Italy). Vascular tissues were equilibrated for 30 min and contracted with 10–5 M NA to develop a maximal response. Preparations were then washed with fresh Krebs' solution, and the equilibration period was allowed to continue for further 30 min. Experiments were carried out on tissues precontracted with NA to 60% of maximal contraction (EC60 = 10–7 M). The endothelium was considered functional when relaxation of precontracted vessels to 10–5 M acetylcholine was at least 80%. Cumulative concentration-response curves for E2, PPT (ER agonist), and DPN (ER agonist) were obtained over the concentration range 10–13 to 10–7 M. To test vehicle effects, cumulative additions of equivalent ethanol dilutions were also performed. Relaxant responses were expressed as percentage of residual NA-induced contraction.
Western Blot Analysis. Cells were washed with phosphate-buffered saline and extracted directly into the lysis buffer as described previously (Idel et al., 2002). The total protein was determined by using Lowry's method (1951). Cell lysate protein was size fractionated on 10% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred onto Hybond enhanced chemiluminescence membranes (Amersham Biosciences Inc. Piscataway, NJ). After blocking with 5% nonfat dry milk (Bio-Rad, Hercules, CA), membranes were hybridized overnight with anti-ER (1:800; Santa Cruz Biotechnology, Inc. Santa Cruz, CA) or anti-ER (1:1000; Calbiochem, San Diego, CA) antibodies, washed, and incubated with horse-radish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The washes were repeated before developing the membrane with enhanced chemiluminescence reagents. The uniformity of protein load and transfer efficiency across the test samples was verified with Ponceau staining. Recombinant ER and ER proteins used as positive controls were from Calbiochem.
Statistical Analysis. All data are expressed as mean ± S.E.M. and represent unpaired data. Concentration-response curves were obtained with software Prism (GraphPad Software Inc., San Diego, CA) and compared by means of two-way analysis of variance (ANOVA). If the curves were statistically different following ANOVA, the treatment affected the response over the tested range of concentrations (Ludbrook, 1994). pD2 values and maximal responses to pharmacological agents were compared by one-way ANOVA followed by Bonferroni's post hoc test, using MINITAB software (Minitab Inc., State College, PA). Values of p < 0.05 were considered significant.
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Results |
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agonist PPT, and the selective ER agonist DPN were obtained in aortic preparations from healthy female rats. None of these compounds affected the tone of nonprecontracted preparations. Acute PPT treatment dose-dependently relaxed NA-precontracted aortic rings by about 30% (pD2 = 10.4 ± 0.4; Emax = 73%; Fig. 1). The relaxant response induced by E2 fully overlapped with that induced by PPT (pD2 = 10.4 ± 0.4; Emax = 76%; Fig. 1). By contrast, the response elicited by DPN was significantly impaired with respect to that elicited by E2 and PPT (pD2 = 9.3 ± 0.6; Emax = 87%; p < 0.05 versus E2 and PPT). Actually, there was no significant response difference between DPN and its vehicle (data not shown). PPT induced a relaxation of the mesenteric artery that was comparable with that of the aorta (pD2 = 10.4 ± 0.3; Emax = 75%; Fig. 1, inset). The method of aorta precontraction influenced the subsequent vasorelaxant response. In fact, precontraction with increasing NA concentrations weakened the relaxant response to PPT (data not shown). When using KCl precontraction, no evidence of specific relaxation by PPT was observed under our experimental conditions (data not shown).
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The relaxant response to PPT was NO- and endothelium-dependent. In fact, the NO-synthase inhibitor L-NAME virtually abolished PPT-induced vasorelaxation in precontracted endothelium-intact aortic rings (pD2 = 9.1 ± 0.3; Emax = 93%; p 
0.001 versus PPT; Fig. 2B). L-NAME treatment also markedly attenuated the acute relaxant response to E2 (pD2 = 9.5 ± 0.4; Emax = 95%; p 0.001 versus E2; Fig. 2A). Similarly, endothelium removal from aortic tissues significantly impaired the response to both PPT and E2 (Emax = 95%; p 0.001 versus PPT and Emax = 92%; p 0.001 versus E2, respectively; Fig. 2).
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0.05 versus PPT and E2; Fig. 3B). The relaxant response to PPT was dependent on interaction with ER, because the pure ER antagonist ICI 182,780 dose-dependently reduced PPT response (Fig. 4). This was also apparent in terms of potency (pD2 PPT alone = 10.7 ± 0.3; PPT + 0.1 µM ICI 182,780 = 10.4 ± 0.3, N.S.; PPT + 1 µM ICI 182,780 = 9.1 ± 0.3, p 0.001 versus PPT alone) and maximal response (PPT + 0.1 µM ICI 182,780 N.S.; PPT + 1 µM ICI 182,780 p 0.01 versus PPT alone).
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Accordingly, we here report for the first time, to the best of our knowledge, that isolated RAECs (Fig. 5A) expressing endothelial NO synthase (Fig. 5B) also expressed detectable amounts of both ER isoforms, as shown by Western blot using recombinant ER and ER as standards for molecular weight and antibody specificity (Fig. 5C).
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Discussion |
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agonists induced acute ER-dependent vasodilation in female rat aorta but lost their efficacy on estrogen deprivation, whereas short-term incubation of aortic rings with selective ER agonists did not elicit any vasoactive effects. We developed a cell culture system of RAECs that allowed us to detect for the first time, to the best of our knowledge, the expression of both ER protein isoforms in these cells. In agreement with previous studies, E2 induced acute relaxation of vascular smooth muscle (Kitazawa et al., 1997). These responses were both endothelium- and nitric oxide-dependent and were significantly inhibited by the pure antiestrogen ICI 182,780, as previously shown in aortic rings from male rats (Bucci et al., 2002), although different mechanisms have been proposed (Shaw et al., 2001). Nongenomic activation of endothelial NO synthase by estrogen via ER has been reported to occur in pulmonary artery (Chen et al., 1999) and bovine aortic endothelial cells (Kim et al., 1999). A role for ER in the modulation of basal NO production ex vivo has also been described in the aorta of ER-knockout mice, although E2 in this study lacked acute vasoactive effects and required long-term or in vivo exposure to be active (Darblade et al., 2002). Our organ bath experiments, however, demonstrate that solely ER mediated the relaxant response to E2 in precontracted aortic preparations from female rats, because the putatively selective ER agonist PPT, but not the putatively selective ER agonist DPN, mimicked the effects of E2. Although the acute relaxation of isolated pressurized rat mesenteric arteries by PPT has been reported to occur only at pharmacological concentrations (Montgomery et al., 2003), our results are novel in that relaxation to PPT occurred 1) not only in NA-precontracted mesenteric artery but also in larger vessels such as the aorta, and 2) at concentrations that are most likely to be achieved in vivo. In the present study, precontraction induced by 60 mM KCl was 3-fold greater than that induced by 0.1 µM NA (data not shown). Therefore, the discrepancies with previously published reports may depend on how and to what extent blood vessels are precontracted as well as on the degree of endothelial functional integrity. On the other hand, DPN was devoid of any significant acute effect on female aortic relaxation, in contrast to the response described in mesenteric arteries from male rats (Montgomery et al., 2003). Acute estrogenic responses seem to be critically dependent on the vessel used. In fact, although PPT is more potent than DPN at producing relaxations in the rat and mouse mesenteric artery, the reverse has been shown to occur in the rat and mouse aorta (Al-Zubair et al., 2004). It is conceivable that ER modulates the vascular relaxation to nonselective agonists such as E2, as shown in studies performed in ER knockout mice (Nilsson et al., 2000), but it seems not to be responsible for relaxation per se. This points to an antagonistic effect of ER on ER as also seen in other contexts (Gustafsson, 2003). Thus, the design of effective, tissue-specific, and safe selective ER agonists may be a promising approach to elicit the vascular beneficial effects of estrogen as shown so far in preclinical studies.
The involvement of ER-mediated pathways into PPT-induced acute vascular relaxation was confirmed because ICI 182,780 dose-dependently abolished this response in E2-treated OVX rats. To substantiate this finding, experiments using the highly selective ER antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole are currently under way. A possible modulation of Ca2+ entry into aortic smooth muscle is unlikely to have accounted for the reported effects of ER agonists on vascular tone, because such modulation usually takes place at agonist concentrations unlikely to be achieved in vivo (Crews and Khalil, 1999; Levin, 2001). Rather, our findings would be consistent with the interaction of PPT (and E2) with membrane ER receptors that 1) activate rapid intracellular signaling and 2) are similar to the described intracellular ER (Russell et al., 2000). This is also in keeping with the recent observation that plasma membrane ERs exist in endothelial cells as functional dimers when bound by steroid ligands, promoting cell-specific signaling pathways (Razandi et al., 2004).
The acute effect of PPT on vascular contractility was recorded in healthy female rats at various stages of estrous cycle. The hormonal status of the animal has been previously shown to be an important determinant of the responsiveness of isolated pressurized rat mesenteric arteries to E2 (Shaw et al., 2001). There may well be an influence of circulating estrogen levels on the magnitude of the acute vasodilator response to ER agonists, because PPT induced greater relaxation of aortic rings obtained from rats 1 week after delivery compared with nonpregnant controls (data not shown). In isolated endothelial cells, E2 induces changes in ER and ER expression that can ultimately modulate hormone effects on vascular endothelium (Ihionkhan et al., 2002). In the absence of circulating estrogen, however, we show that the selective ER agonist as well as E2 completely lost the ability to induce acute vascular relaxation in aortic tissues from untreated OVX rats, which was restored by an E2 replacement protocol yielding high physiological serum E2 levels (Watson et al., 1986). Conversely, DPN still failed to affect vascular reactivity after either ovariectomy or E2 replacement. Therefore, ER function is critically sensitive to E2 deprivation as observed in OVX rats. Ovariectomy has been shown not to modulate significantly ER expression in rat aorta (Mohamed and Abdel-Rahman, 2000), but it may have led to loss of function for ER that could be restored upon administration of its physiological ligand. There are indications that 4 weeks of treatment with another selective ER agonist, Cpd1471, starting 1 day after ovariectomy, improves endothelial dysfunction in OVX spontaneously hypertensive rats by restoring endothelial NO synthase protein levels (Widder et al., 2003). Consequently, it can be speculated that the success of any pharmacological treatment with estrogenic compounds after menopause relies on the duration of E2 deprivation. To verify this hypothesis, we are currently investigating whether E2 replacement as carried out in the present study affects ER expression in isolated RAECs and retains the ability to restore acute vasomotor responses to ER agonists after long-term (3–6-month) ovariectomy.
In conclusion, this study showed that selective ER agonists, unlike selective ER agonists, induced acute vascular relaxation in the aorta from intact female or E2-replaced OVX rats by interacting with ER rather than with ER, both of which are expressed in isolated RAECs. Such an activity, however, was undetectable in preparations from untreated OVX rats. These findings may have implications for selective targeting of ER isoforms in treating vascular disease.
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Footnotes |
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ABBREVIATIONS: ER, estrogen receptor; E2, 17-estradiol; PPT, 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl) tris-phenol; DPN, 2,3-bis(4-hydroxyphenyl)-propionitrile; OVX, ovariectomized; NA, noradrenaline; L-NAME, N-nitro-L-arginine methyl ester; ICI 182,780, 7,17-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol; RAEC, rat aortic endothelial cell; ANOVA, analysis of variance.
Address correspondence to: Dr. Andrea Cignarella, Department of Pharmacological Sciences, University of Milan, via Balzaretti 9, I-20133 Milan, Italy. E-mail: andrea.cignarella{at}unimi.it
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