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(Circulation Research. 1997;81:242-248.)
© 1997 American Heart Association, Inc.

Articles

Mechanism of Vascular Smooth Muscle Relaxation by Estrogen in Depolarized Rat and Mouse Aorta

Role of Nuclear Estrogen Receptor and Ca²⁺ Uptake

Ana D. Freay, Sylvia W. Curtis, Kenneth S. Korach, , Gabor M. Rubanyi

From the Cardiovascular Research Department (A.D.F., G.M.R.), Berlex Biosciences, Richmond, Calif, and the Laboratory of Reproductive and Developmental Toxicology (S.W.C., K.S.K.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC.

Correspondence to Gabor M. Rubanyi, MD, PhD, Berlex Biosciences, Cardiovascular Research, 15049 San Pablo Ave, Richmond, CA 94804. E-mail rubanyi_gabor{at}berlex.com

	Abstract

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Abstract 17ß-Estradiol induces vasodilation in vitro and in vivo, which has been suggested to contribute to the cardiovascular protection by this ovarian steroid hormone. However, the exact mechanism of vasorelaxation by estrogens remains to be elucidated. In this study, we analyzed the potential role of genomic mechanisms involving the nuclear estrogen receptor and inhibition of entry of extracellular Ca²⁺ in 17ß-estradiol–induced vasorelaxation in depolarized aortic rings, isolated from male and female rats and male mice. In both male and female rat aortic rings without endothelium and in intact male mouse aortic rings treated with N^G-nitro-L-arginine, 17ß-estradiol caused dose-dependent (0.3 to 30 µmol/L) relaxation of contraction evoked by high-K⁺ depolarization (30 and 45 mmol/L KCl, respectively). The estrogen receptor antagonist ICI 164384 had no effect on 17ß-estradiol–induced relaxations. ¹²⁵I-17ß-estradiol binding studies showed the presence of high-affinity cytosolic-nuclear estrogen receptors in control male mouse aortas. Comparable relaxations of aortic rings isolated from control and estrogen receptor–deficient transgenic mice provided direct evidence that the nuclear estrogen receptor is not involved in this response. 17ß-Estradiol–induced relaxation of rat aortic rings could not be prevented by cycloheximide or actinomycin D, suggesting that the response was not mediated by de novo protein synthesis or gene transcription. In rat aortic rings, 17ß-estradiol inhibited the increase of ⁴⁵Ca uptake by 30 mmol/L KCl at concentrations (10 and 30 µmol/L) that caused vasorelaxation in the same tissue, suggesting that inhibition of Ca²⁺ entry contributes to the response. 17-Estradiol was less effective, and estrone was devoid of vasorelaxing activity. Vasorelaxation by estrogens in female and male rat aortas was similar, indicating no gender difference in vascular responses under these conditions.

Key Words: 17ß-estradiol • 17-estradiol • estrone • diethylstilbestrol • mice, estrogen receptor–deficient

	Introduction

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Hormone replacement therapy (using estrogen alone or in combination with progestins) of postmenopausal women significantly reduces morbidity and mortality due to cardiovascular diseases.^{1 2} Although the beneficial effect of estrogens on plasma lipoproteins (ie, lowering low-density lipoprotein and increasing high-density lipoprotein cholesterol) contributes to cardiovascular protection,^{1 3} it does not fully account for the protective effect.^{2 4 5 6} Therefore, it was postulated that direct effects on the cardiovascular system also contribute to the protection against atherosclerosis. Indeed, estrogen receptors were localized in the blood vessels and heart.^{7 8 9 10 11} Furthermore, 17ß-estradiol was shown to affect cardiovascular function in animals and humans. Estrogens modulate vascular tone via at least two mechanisms. After chronic treatment of animals, they increase the production of the endothelium-derived vasodilator NO,^{12 13 14 15 16 17 18} and they also relax vascular smooth muscle directly.^{19 20 21} The rapid vasorelaxation by relatively high (pharmacological) concentrations of estrogen was suggested to be due to nongenomic mechanisms²² (ie, not modified by inhibitors of gene transcription or protein synthesis as seen in the central nervous system^{23 24} ). However, the presence of functional estrogen receptors in vascular smooth muscle cells of the rat aorta^{25 26} and the fact that both the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor actinomycin D inhibited the smooth muscle relaxing effect of estradiol in the isolated rat uterus²⁷ have suggested that genomic mechanisms may also contribute to the response. Several studies have suggested that estrogen-induced direct vasorelaxation is due to inhibition of the entry of extracellular Ca²⁺ into vascular smooth muscle cells.^{20 21} The present study was designed to analyze the mechanism of direct vascular smooth muscle–relaxing action of 17ß-estradiol in depolarized rings of rat and mouse aortas. Studies were performed to determine the effect of vasorelaxing concentrations of 17ß-estradiol on inhibiting the influx of extracellular Ca²⁺. The involvement of genomic mechanisms²⁸ was analyzed using inhibitors of gene transcription (actinomycin D) and de novo protein synthesis (cycloheximide). Assessment of the nuclear estrogen receptor participation was done by using the estrogen receptor–specific antagonist ICI 164384. Aortic rings isolated from ERKO mice²⁹ for the first time allowed direct evaluation of the role of the classical estrogen receptor in the response.

	Materials and Methods

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Isolated Rat Aorta
Male Sprague-Dawley rats (150 to 250 g body weight) were killed by inhalation of CO₂. The thoracic aorta was dissected, cleaned of loose adipose and connective tissue, and cut into 2- to 3-mm-wide rings. The endothelium was removed mechanically from the intimal surface of each aortic ring by inserting a piece of wire through the lumen. Aortic rings were mounted between a force displacement transducer (model FT03C, Grass Instrument Co) and a rigid support for measurement of isometric force, which was displayed on a recorder (model TA500, Gould Inc). Tissues were incubated in 10-mL organ baths (kindly made available by A.A. Pesce, Kennett Square, Pa) containing warmed (37°C) oxygenated (95% O₂/5% CO₂) KHS of the following composition (mmol/L): NaCl 118, KCl 4.8, CaCl₂ 2.5, MgSO₄ 1.2, NaHCO₃ 24, glucose 11.1, tetrasodium EDTA 0.03, and ibuprofen 0.01 (pH 7.4). Passive tension was adjusted over a 30-minute equilibration period to 1 g, and the KHS was exchanged at 10-minute intervals. The aortic rings were then contracted with phenylephrine (0.3 µmol/L), and the absence of endothelium was tested by acetylcholine (1 µmol/L); segments showing >10% relaxation were discarded. After washout of phenylephrine and acetylcholine, the aortic rings were allowed to equilibrate for 20 minutes and were then contracted again with 30 mmol/L KCl. When a stable contraction was obtained, one concentration of 17ß-estradiol (0.3, 1, 3, 10, or 30 µmol/L) was added to the tissue bath. Tissues were preincubated with cycloheximide (10 µmol/L), actinomycin D (10 µmol/L), or the estrogen receptor antagonist ICI 164384 (30 µmol/L) for 45 minutes before KCl contraction and 17ß-estradiol relaxation. In another series of experiments, the effects of various estrogens were tested on aortic rings (contracted with 30 mmol/L KCl) by adding increasing concentrations (0.3 to 30 µmol/L) of the compounds in a cumulative manner. Tissues were exposed to each dose for 15 minutes before the next dose was added.

Isolated Mouse Aorta
Aortas were isolated from male control C57Bl/6J mice and also from male ERKO mice.²⁹ The mice were killed in a CO₂ chamber. The thoracic aorta was rapidly removed into warm (37°C) KHS (pH 7.4 when aerated with 95% O₂ and 5% CO₂) containing ibuprofen (10 µmol/L). The aortas were cleaned of fat and connective tissue and cut into 0.5-mm-long rings. The rings were transferred into an organ bath (37°C, KHS) and suspended for isometric recording of tension. Rings were equilibrated for 90 minutes under minimal resting tension and then stretched to optimal passive tension (500 mg). Since mechanical removal of the endothelium from the thin-walled mouse aortic rings was not possible, endothelial NO production was prevented by treatment with LNNA (100 µmol/L). The effective blockade of endothelial NO production was tested in each ring by the absence of acetylcholine-induced relaxation. Rings exhibiting >10% acetylcholine relaxation were excluded from the study. After tissues reached steady state contraction to 45 mmol/L KCl, 17ß-estradiol and other estrogens were added to the tissue bath at increasing doses (0.3 to 30 µmol/L) in a cumulative manner at 15-minute intervals. In separate experiments (n=4), ICI 164384 (30 µmol/L) was added 30 minutes before 17ß-estradiol in 30 mmol/L KCl-contracted ERKO mouse aortic rings.

Estrogen Receptor Binding Assay in Mouse Aorta
Aortas from 50 C57B1/6J male mice were isolated and immediately frozen in liquid nitrogen. The aortas were homogenized in TEGM buffer (10 mmol/L Tris, 1.5 mmol/L EDTA, 10% glycerol, 3 mmol/L MgCl₂, and 3 mmol/L EGTA) with protease inhibitors (50 µg/mL each of leupeptin, antipain, soybean trypsin inhibitor, and chymostatin) using a Polytron homogenizer (Brinkman Instruments). The homogenate was filtered through Nitex (Tetco Inc) and then centrifuged to pellet nuclei at 2500g. The supernatant was spun at 5000g for 50 minutes to clear the cytosol, which was then used in the binding assay. Nuclei were washed twice and resuspended in homogenization buffer for assay. ¹²⁵I-Estradiol (New England Nuclear) binding was determined in three different groups of cytosolic and nuclear preparations prepared from 15-15 different aortas by using methods previously described,^{30 31 32} with the following modification: instead of dissolving and counting the hydroxyapatite pellet in scintillation fluid, we placed tubes containing drained hydroxyapatite pellets in a gamma counter. Data were analyzed using Accufit Sturation Two Site software (Lundon Software), normalized for cellular DNA content, and expressed as mean±SEM (n=3).

Estrogen Receptor Binding Assay in Rat Uterus
Estrogen receptor binding affinity of the compounds used was determined in estrogen receptor preparations of rat uterus and expressed as CF. Estrogen receptor–containing cell lysate was incubated with [³H]estradiol in the presence of the test compounds (concentration range, 1 nmol/L to 1 µmol/L) or unlabeled estradiol as a reference. Unbound [³H]estradiol was then extracted by incubation with dextran-coated charcoal for 5 minutes. The charcoal was separated by high-speed centrifugation. Receptor-bound [³H]estradiol was determined by liquid scintillation counting. Competition curves were obtained for the compounds and for the reference estradiol. The CF is calculated as follows: the concentration of test compound needed to cause 50% displacement of [³H]estradiol from the receptor is divided by the concentration of unlabeled estradiol needed to cause the same displacement.

⁴⁵Ca-Uptake Studies
The method used to measure ⁴⁵Ca uptake was essentially the same as described by Aaronson.³³ Male Sprague-Dawley rats (150 to 250 g) were killed as described above. The thoracic aortas were rapidly removed from the animals and cleaned of fat in warm (37°C) PSS of the following composition (mmol/L): NaCl 140, KCl 5, MgCl₂ 1, glucose 10, CaCl₂ 1.5, and sodium HEPES 5. The aorta was cut into rings of 5 mm length, and the endothelium was removed and mounted unstretched on stainless steel hooks. Rings were allowed to recover for 90 minutes in PSS at 37°C. Tissues were then transferred to culture tubes filled with PSS solution (4 mL) containing 10 µCi/mL ⁴⁵Ca. After 2-minute labeling, tissues were transferred to ice-cold 2 mmol/L EGTA-PSS (10 mL) solution for 30 minutes. At the end of each experiment, tissues were blotted briefly and weighed. Tissues were placed overnight in 2.5 mL of a 5 mmol/L EDTA solution to disperse tissue ⁴⁵Ca and were counted, along with samples of labeling solutions, in a scintillation counter (Beckman LS3801).

Lipid Solubility
The lipid solubility of the estrogenic compounds used in the present study was determined by calculating log P (n-octanol/water partition coefficient) values using the computer program Polaris 3.0 (Molecular Symulations Inc).

Drugs
Cycloheximide, actinomycin D, 17ß-estradiol, 17-estradiol, estrone, DES, and potassium chloride were obtained from Sigma Chemical Co. ICI 164384 was obtained from Schering AG. All estrogenic compounds were dissolved in DMSO. The highest final concentration of DMSO in the organ bath was 0.2% (vol/vol), which on its own (vehicle control studies) caused no smooth muscle relaxation or change in ⁴⁵Ca uptake.

Calculations and Statistical Analysis
Relaxation of aortic rings is expressed as a percentage of the initial contraction to KCl. ⁴⁵Ca content is expressed as micromoles ⁴⁵Ca per kilogram tissue wet weight. Results are expressed as the mean±SEM. The number (n) of experiments represents the number of aortic rings isolated from different animals (except for receptor binding studies in mouse aortas, where n represent aortas pooled from 15 animals). Statistical comparisons were performed using a one-way ANOVA for repeated measures, followed by the Student-Newman-Keuls test for multiple comparisons. A value of P<.05 was considered statistically significant.

	Results

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Relaxation of Depolarized Male Rat Aortic Rings by 17ß-Estradiol
The original trace in Fig 1 shows relaxation of an aortic ring contracted with 30 mmol/L KCl by 10 µmol/L 17ß-estradiol. The ovarian sex steroid hormone initiated the response rapidly, and the relaxation reached a steady level after 45 minutes. The extent of relaxation increased in a dose-dependent manner in the concentration range of 0.3 to 30 µmol/L (Fig 2, control curve).

View larger version (5K): [in this window] [in a new window]   Figure 1. Original trace showing the time course of vascular smooth muscle relaxation by 17ß-estradiol (10 µmol/L) in an endothelium-denuded male rat aortic ring contracted by 30 mmol/L KCl.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of actinomycin D (10 µmol/L) (), cycloheximide (10 µmol/L) (), and ICI 164384 (30 µmol/L) () on 17ß-estradiol–induced vasorelaxation (control) () in 30 mmol/L KCl–depolarized male rat aortic rings without endothelium. Results are expressed as percent change of the initial KCl contraction and are shown as mean±SEM (n=4). +Statistically significant difference from control. *Statistically significant difference for the effect of 17ß-estradiol vs vehicle control (not shown).

In order to analyze the involvement of the nuclear estrogen receptor, gene transcription, and/or de novo protein synthesis in the response, the effects of pretreatment (45 minutes) with the protein synthesis inhibitor cycloxheximide (10 µmol/L), the gene transcription inhibitor actinomycin D (10 µmol/L), and the selective estrogen receptor antagonist ICI 164384 (30 µmol/L) were tested on vasorelaxation induced by 17ß-estradiol. None of these inhibitors suppressed the relaxation induced by 17ß-estradiol in depolarized rat aortas (Fig 2). Cycloheximide pretreatment significantly augmented relaxations evoked by 1, 10, and 30 µmol/L 17ß-estradiol.

Effect of 17ß-Estradiol on ⁴⁵Ca Uptake in Depolarized Male Rat Aorta
Exposure to 30 mmol/L KCl caused significant increase in cellular ⁴⁵Ca content in endothelium-denuded rings of rat aortas (Fig 3). The dihydropyridine Ca²⁺ channel antagonist nifedipine (1 µmol/L) prevented KCl-induced ⁴⁵Ca uptake. 17ß-Estradiol, at concentrations that caused significant vascular smooth muscle relaxation (10 and 30 µmol/L) in the same tissue (see Figs 1 and 2), inhibited KCl-induced ⁴⁵Ca uptake in a dose-dependent manner.

View larger version (12K): [in this window] [in a new window]   Figure 3. Effect of nifedipine (NIF, 1 µmol/L) and 17ß-estradiol (E2, 10 and 30 µmol/L ) on 30 mmol/L KCl–induced 45Ca uptake in endothelium-denuded male rat aortic rings. The results are expressed as micromoles Ca per kilogram tissue wet weight and are shown as mean±SEM (n=4). *Statistically significant difference from control (C). +Statistically significant difference from KCl. #Statistically significant difference between 10 and 30 µmol/L 17ß-estradiol.

Relaxation of Depolarized Male and Female Rat Aortic Rings by 17ß-Estradiol, 17-Estradiol, Estrone, and DES
The effect of 17ß-estradiol was compared with that of the weaker estrogen, 17-estradiol (CF, 46), the more potent synthetic estrogen, DES (CF, 0.3), and the natural estradiol metabolite, estrone (CF, 7.4). The estrogens were tested at cumulatively increasing concentrations (0.3, 1, 3, 10, and 30 µmol/L) on both male and female rat aortic rings contracted with 30 mmol/L KCl. The estrogenic compounds showed dose-dependent relaxation of varying degrees in both male and female rat aortic rings (Fig 4). 17-Estradiol was less effective than DES or 17ß-estradiol, whereas estrone caused no vasorelaxation in the concentration range studied in both male and female aortic rings. Differences in lipid solubility cannot be the cause of these results, since 17ß- and 17-estradiol, estrone, and DES had very similar octanol/water partition coefficients (log P values, 4.32, 4.28, 4.32, and 4.25, respectively). The results in female rat aortas (Fig 4b) show that the extent of vasorelaxation by these compounds was not statistically different and that the rank order was similar to that observed in male rat aortic preparations (Fig 4a).

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of increasing concentrations (0.3 to 30 µmol/L) of 17ß-estradiol (), 17-estradiol (), estrone (), and DES () on 30 mmol/L KCl–induced contraction in endothelium-denuded male (a) and female (b) rat aortic rings. Vehicle control () was performed by adding DMSO only (cumulative final concentration, 0.2% [vol/vol]). Results are expressed as percent change of the initial KCl contraction and are shown as mean±SEM (n=4). +Statistically significant difference from vehicle control. *Statistically significant difference from 17ß-estradiol group.

¹²⁵I-17ß-Estradiol Binding in Mouse Aorta
Scatchard plot analysis of binding data showed the presence of high-affinity 17ß-estradiol binding sites in both the cytosol (K_d, 2.9±0.5 nmol/L; B_max, 119±25 fmol/100 µg DNA; n=3) and nucleus (K_d, 1.3±0.3 nmol/L; B_max, 142±27 fmol/100 µg DNA; n=3) of aortic tissue from male control mice. Fig 5 shows a representative saturation binding curve (left) and Scatchard plot (right) of ¹²⁵I-17ß-estradiol binding to the cytosolic fraction, prepared from 15 aortic tissues.

View larger version (11K): [in this window] [in a new window]   Figure 5. Saturation binding curve (a) and Scatchard plot (b) of 125I-17ß-estradiol binding to the cytosolic fraction of aortic tissue from male rats. NS indicates nonspecific.

Vasorelaxation in Depolarized Aortic Rings From Control and ERKO Male Mice
In depolarized (45 mmol/L KCl) aortic rings isolated from control male mice, DES, 17ß-estradiol, and 17-estradiol showed concentration-dependent relaxation (Fig 6a). DES was more effective than 17ß-estradiol in mouse (Fig 6a) but not in rat (Fig 4a) aortas, and 17-estradiol caused significantly less relaxation than DES or 17ß-estradiol in both male rat (Fig 4a) and mouse (Fig 6a) aortas. Aortic rings isolated from ERKO mice showed dose-dependent relaxation to the three estrogens (Fig 6b) similar to that observed in control mouse aortas (Fig 6a). Pretreatment of ERKO mouse aortic rings with ICI 164384 (30 µmol/L) had no significant effect on 17ß-estradiol–induced relaxation (n=4, not shown).

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of increasing concentrations (0.3 to 30 µmol/L) of 17ß-estradiol (), 17-estradiol (), and DES () on 45 mmol/L KCl–induced contraction in aortic rings isolated from wild type (control) (a) and ERKO (b) male mice. The experiments were performed on intact rings treated with LNNA (10 µmol/L). Results are expressed as percent change of the initial KCl contraction and are shown as mean±SEM (n=4). *Statistically significant difference from 17ß-estradiol group. +Statistically significant difference from vehicle control ().

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study shows that 17ß-estradiol relaxes high-KCl–induced contraction of isolated male and female rat and male mouse aortic rings in a concentration-dependent manner. This observation is in good agreement with earlier demonstrations of 17ß-estradiol–induced vasorelaxation in other types of isolated blood vessels^{19 34} and vasodilation in isolated perfused hearts^{35 36} and in intact animals.³⁷ The relative high concentrations of 17ß-estradiol required to elicit vasorelaxation in isolated large arteries in the present and several earlier studies^{19 20 21 27 37} suggest that experiments performed under these conditions are predominantly of pharmacological significance. However, they offer an opportunity to study the mechanism of vascular actions of estrogens, which is very difficult under more physiological in vivo conditions.

The vasorelaxation by 17ß-estradiol was not mediated by prostacyclin (the experiments were performed in the presence of the cyclooxygenase inhibitor ibuprofen) or by endothelium-derived NO, because the aortic rings were either denuded of endothelium (rat) or treated with LNNA (mouse). Thus, the effect must be due to direct action of the ovarian steroid hormone on vascular smooth muscle cells under these experimental conditions.

Although several potential mechanisms were proposed,^{38 39 40 41} the exact mechanism by which estrogen modulates vascular tone is not fully understood.

Role of Inhibition of Ca²⁺ Entry Into Smooth Muscle
17ß-Estradiol is a more potent vasorelaxant in high K⁺–depolarized vascular preparations than in those contracted with various receptor agonists.³⁴ Therefore, it was suggested that the ovarian steroid hormone relaxes smooth muscle predominantly by inhibiting the entry of Ca²⁺ into smooth muscle cells.^{20 21} Indeed, the present study showed that similar to the dihydropyridine Ca²⁺ antagonist nifedipine, 17ß-estradiol prevented high K⁺–induced ⁴⁵Ca uptake in rat aortic rings in a concentration-dependent fashion, which correlated well with the vasorelaxation observed in this tissue. These data suggest that inhibition of Ca²⁺ entry into depolarized vascular smooth muscle cells may be an important component of 17ß-estradiol–induced vasorelaxation.

Role of Genomic Mechanisms and the Nuclear Estrogen Receptor
It is not clear whether the inhibitory effect of 17ß-estradiol on vascular smooth muscle tone is mediated by the nuclear estrogen receptor. Specific high-affinity cytosolic nuclear binding sites for 17ß-estradiol have been found earlier in rat aortic vascular smooth muscle cells.^{25 26} The present study for the first time demonstrated the existence of high-affinity cytosolic-nuclear estrogen binding sites in the mouse aorta. The aorta of control mice expresses the wild-type ER gene, whereas the ERKO aorta shows only the disrupted ER gene products, which are functionally inactive.⁴² A second nuclear estrogen receptor termed ERß has recently been cloned from rat prostate and ovary.⁴³ It is still uncertain whether this gene is expressed or the receptor protein functional in the aortas of ERKO mice.

Several lines of evidence argue against the possibility that smooth muscle relaxation observed in isolated rat and mouse aortic rings is mediated by genomic mechanisms involving the nuclear estrogen receptors: (1) the concentrations of 17ß-estradiol required to evoke smooth muscle relaxation are several orders of magnitude higher than those required for genomic actions^{24 28} ; (2) the rapid onset of action of 17ß-estradiol is inconsistent with a response requiring gene transcription; and (3) the selective nuclear estrogen receptor antagonist, ICI 164384, and inhibitors of gene transcription (actinomycin D) and de novo protein synthesis (cycloheximide) did not suppress vascular smooth muscle relaxation by 17ß-estradiol. The most direct proof for the conclusion that the nuclear estrogen receptor is not involved in estrogen-induced vasorelaxation was provided by the present studies on aortic rings isolated from ERKO mice: the relaxation of depolarized aortic rings to 17ß-estradiol, 17-estradiol, and DES were similar in aortic preparations isolated from control and ERKO mice. Theoretically the ERß receptor may play a role in mediating vasorelaxations in the ERKO mouse aorta. However, the lack of inhibition of 17ß-estradiol–induced relaxation by ICI 164384 in the ERKO mouse aorta rules out the involvement of ERß receptors, because this antagonist effectively blocks ERß-mediated estrogen responses as well.^{43 44}

It is probable that these nongenomic rapid responses are mediated by interaction with the cell membrane and consequent changes in membrane function (eg, ion channel properties). Although a surface membrane "estrogen" receptor has not been identified in vascular smooth muscle cells, previous studies have shown that several cells may bear nongenomic cell surface binding sites (receptors) for 17ß-estradiol.^{45 46} Because of the demonstrated inhibition of high K⁺–induced ⁴⁵Ca uptake, it is also possible that 17ß-estradiol binds to the voltage-dependent Ca²⁺ channel in smooth muscle membrane. Although the present study was not designed to test this possibility, studies on human bladder and myometrium ruled out direct interaction with high-affinity dihydropyridine binding sites in L-type Ca²⁺ channels.⁴⁷

Structure-Activity Relationship
Analysis of the structure-activity relationship led to several important novel observations. It suggested that the smooth muscle relaxant effect of 17ß-estradiol may not be due to nonspecific effects related to its basic steroidal structure, because the nonsteroidal estrogen, DES, exerted potent smooth muscle relaxing activity, whereas the natural steroidal metabolite, estrone, had no effect on smooth muscle contraction.

The other novel observation of the present study was that C17 substitution has a profound effect on the vasorelaxing efficacy of 17ß-estradiol. Substitution of 17ß-OH with 17-OH (17-estradiol) significantly lowered, whereas substitution with a keto group at the 17 position (estrone) prevented, vasorelaxing efficacy in male and female rat aortas. This finding is in good agreement with a recent report showing a lack of Ca²⁺-antagonistic properties by estrone and 17-estradiol in cultured human vascular smooth muscle cells.⁴⁸

In summary, estrogen-induced vasorelaxation in the depolarized rat aorta is not mediated by genomic mechanisms or the nuclear estrogen receptor but is associated with inhibition of Ca²⁺ entry into vascular smooth muscle cells. Using the estrogen receptor–deficient mouse, the present study provided the first direct evidence that the classical nuclear estrogen receptor (ER) is not involved in estrogen-induced rapid vasorelaxation. The analysis of the structure-activity relationship led to the novel observation that the stereoisomeric configuration of the -OH group or its substitution with a keto group at the C17 position causes significant reduction in vascular smooth muscle relaxation.

	Selected Abbreviations and Acronyms

 

CF = competition factor DES = diethylstilbestrol DMSO = dimethyl sulfoxide ER, ERß = estrogen receptor alpha and beta ERKO = estrogen receptor–deficient transgenic KHS = Krebs-Henseleit solution LNNA = NG-nitro-L-arginine PSS = physiological saline solution

	Acknowledgments

 
We express special thanks to Dr Karl-Heinrich Fritzemeier (Schering AG, Berlin, Germany) for the performance of rat uterine estrogen receptor binding studies.

Received January 7, 1997; accepted May 1, 1997.

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