The effects of estrogen on the α2-adrenergic receptor subtypes in rat uterine function in late pregnancy in vitro

Aim To assess the effect of 17β-estradiol pretreatment on the function and expression of α2- adrenergic receptors (ARs) subtypes in late pregnancy in rats. Methods Sprague-Dawley SPD rats (n = 37) were treated with 17β-estradiol for 4 days starting from the 18th day of pregnancy. The myometrial expression of the α2-AR subtypes was determined by real time polymerase chain reaction and Western blot analysis. In vitro contractions were stimulated with (-)-noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (α2A), ARC 239 (α2B/C), and spiroxatrine (α2A). The cyclic adenosine monophosphate (cAMP) accumulation was also measured. The activated G-protein level was investigated by guanosine 5′-O-[gamma-thio]triphosphate (GTPγS) binding assay. Results 17β-estradiol pretreatment decreased the contractile effect of (-)-noradrenaline via the α2-ARs, and abolished the contractile effect via the α2B-ARs. All the α2-AR subtypes’ mRNA was significantly decreased. 17β-estradiol pretreatment significantly increased the myometrial cAMP level in the presence of BRL 44408 (P = 0.001), ARC 239 (P = 0.007), and spiroxatrine (P = 0.045), but did not modify it in the presence of spiroxatrine + BRL 44408 combination (P = 0.073). It also inhibited the G-protein-activating effect of (-)-noradrenaline by 25% in the presence of BRL 44408 + spiroxatrine combination. Conclusions The expression of the α2-AR subtypes is sensitive to 17β-estradiol, which decreases the contractile response of (-)-noradrenaline via the α2B-AR subtype, and might cause changes in G-protein signaling pathway. Estrogen dysregulation may be responsible for preterm labor or uterine inertia via the α2-ARs.

In spite of the numerous attempts to explore it, the exact action mechanism and risk of preterm birth still remains one of the biggest challenges in obstetrics and gynecology and a major contributor to perinatal mortality and morbidity, affecting around 9% of births in developed countries (1)(2)(3)(4). On the other hand, weak contractions and poor labor outcomes also represent a problem mainly among obese women, increasing the number of cesarean deliveries (5).
Uterine contractility is regulated by several factors, such as the adrenergic system (6) and female sexual hormones (7,8). Progesterone was demonstrated to increase the synthesis of β 2 -ARs during pregnancy (9)(10)(11) and the number of activated G-proteins (12,13), which is why it can be combined with β 2 -AR agonists in threatening preterm labor. Myometrial α 1 -AR expression is influenced by female sexual steroid hormones, mainly estrogens. 17β-estradiol decreases the expression of the α 1A -ARs, but does not influence the expression of α 1D -ARs (14). However, the effect of estrogens on the myometrial α 2 -AR subtypes is unknown. Considering the fact that estrogens play a major role in myometrial contractions during human parturition (15,16), it is important to know if they have a direct influence on the α 2 -AR subtypes, which are also involved in the mechanism of uterine contractions (17).
The α 2 -ARs have been divided into (18,19) α 2A , α 2B , and α 2C subtypes. All three receptor subtypes are coupled to the pertussis toxin-sensitive G i -protein α-subunit (20) and decrease the activity of adenylyl cyclase (AC) and voltagegated Ca 2+ currents, at the same time activating the receptor-operated K + currents (21). The stimulation of these receptors leads to presynaptic feedback inhibition of (-)-noradrenaline release on the adrenergic neurons (18), and mediates a variety of cell functions, such as vasoconstriction, increased blood pressure, and nociception. Furthermore, all three α 2 -AR subtypes were identified in both pregnant and non-pregnant myometrium and were shown to take part in both increased and decreased myometrial contractions (22,23). Under certain circumstances, α 2 -ARs can couple not only to G i -proteins but to G s -proteins, resulting in the activation of AC (24). On the other hand, pregnancy has been proved to induce a change in the G i /G s -activating property of the α 2 -ARs in rats, resulting in a differential regulation of myometrial AC activity in mid-pregnancy vs term (25). The α 2B -ARs were shown to predominate and mediate contraction in last-day-pregnant animals by decreasing the intracellular cAMP level, while α 2A -and α 2C -ARs mediate only weak contractions by increasing the cAMP level, which can be regarded as relaxation as they are compared with the effect of (-)-noradrenaline (23).
Since female sexual steroid hormones play an important role in the regulation of the adrenergic receptor system (26), the effect of estrogen on different α 2 -AR subtypes has been investigated. The mRNA expression of the α 2A -ARs in the spinal cord was increased after estrogen pretreatment (27), which could contribute to the higher prevalence of pain syndromes in women. On the other hand, estrogen was shown to increase the smooth muscle expression of α 2C -ARs and therefore the cold-induced constriction of cutaneous arteries (28). In addition, it was shown to stimulate the (-)-noradrenaline release in the hypothalamus due to the decreased coupling of the α 2 -adrenoceptors to G protein (29).
Since there are no available data on the effects of 17βestradiol on the myometrial functions of different α 2 -AR subtypes, the aim of this study was to clarify the changes in expression and function of the α 2A -, α 2B -, and α 2C -AR subtypes after 17β-estradiol pretreatment on the last day of pregnancy in rats by using RT-PCR and Western blot analysis. Since the changes in the intracellular cAMP are crucial in the control of smooth muscle contractions and relaxations, our further aim was to measure the cAMP release after 17β-estradiol pretreatment in the presence of the subtype-specific α 2 -AR antagonists. We also investigated the changes in the G-protein activation of α 2 -ARs using GTPγS binding assay.

MATERiAlS AnD METHoDS
The animal experimentation was carried out with the approval of the Hungarian Ethics Committee for Animal Research (permission number: IV/198/2013). The animals were treated in accordance with the European Communities Council Directives (86/609/ECC) and the Hungarian Act for the Protection of Animals in Research (XXVIII. tv. 32. §).

Housing and handling of the animals
Sprague-Dawley rats were obtained from the INNOVO Ltd (Gödöllő, Hungary) and were housed under controlled temperature (20-23°C), in humidity (40%-60%) and light (12 h light/dark regime) regulated rooms. The animals were fed standard rodent pellet diet (INNOVO Ltd, Isaszeg, Hungary), with tap water available ad libitum.

Mating of the animals
Mature female (180-200 g, n = 58) and male (240-260 g, n = 12) Sprague-Dawley rats were mated in a special mating cage with a time-controlled electrically movable metal door separating the rooms for male and female animals. Since rats are usually active at night, the door was opened before dawn. Within 4-5 hours after the possibility of mating, female rats with the presence of copulation plug or a sperm-positive vaginal smear (search was performed under under a microscope at a magnification of 1200 times) were separated. The day of copulation was considered as the first day of pregnancy.

In vivo sexual hormone treatments of the rats
The 17β-estradiol (Sigma Aldrich, Budapest, Hungary) pretreatment of the pregnant animals was started on the day 18 of pregnancy. The compound was dissolved in olive oil. The animals were injected subcutaneously with 5 μg/kg of 17β-estradiol once a day for 4 days (30). On the day 22, the uterine samples were collected and the contractility and molecular pharmacological studies were carried out.

RT-PCR studies
Tissue isolation: Rats (250-300 g) were sacrificed by CO 2 asphyxiation. Newborn rats were sacrificed by immediate cervical dislocation. The uterine tissues from pregnant animals (n = 5 in each experiment) (tissue between two implantation sites) were rapidly removed and placed in RNAlater Solution (Sigma-Aldrich). The tissues were frozen in liquid nitrogen and stored at -70°C until total RNA extraction.
Total RNA preparation from tissue: Total cellular RNA was isolated by extraction with guanidinium thiocyanate-acidphenol-chloroform according to Chomczynski and Sacchi (31). After precipitation with isopropanol, the RNA was washed with 75% ethanol and then re-suspended in diethyl pyrocarbonate-treated water. RNA purity was controlled at an optical density of 260/280 nm with BioSpec Nano (Shimadzu, Japan); all samples exhibited an absorbance ratio in the range 1.6-2.0. RNA quality and integrity were assessed by agarose gel electrophoresis.
Reverse transcription and amplification of the PCR products was performed by using the TaqMan RNA-to-CTTM 1-Step Kit (Life Technologies, Budapest, Hungary) and the ABI StepOne Real-Time cycler. RT-PCR amplifications were performed as follows: 48°C for 15 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, and 60°C for 1 min. The generation of specific PCR products was confirmed by melting curve analysis. Table 1 shows the assay IDs for the used primers. The amplification of β-actin served as an internal control. All samples were run in triplicates. The fluorescence intensities of the probes were plotted against PCR cycle numbers. The amplification cycle displaying the first significant increase in the fluorescence signal was defined as the threshold cycle (Ct).
Western blot analysis 20 μg of protein per well was subjected to electrophoresis on 4%-12% NuPAGE Bis-Tris Gel in XCell SureLock Mini-Cell Units (Life Technologies) (n = 5 for each α 2 -AR subtype antagonists). Proteins were transferred from gels to nitrocellulose membranes, using the iBlot Gel Transfer System (Life Technologies). The antibody binding was detected with the WesternBreeze Chromogenic Western blot immundetection kit (Life Technologies). The blots were incubated on a shaker with α 2A -AR, α 2B -AR, α 2C -AR, and β-actin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:200) in the blocking buffer. Images were captured using the EDAS290 imaging system (Csertex Ltd, Budapest, Hungary), and the optical density of each immunoreactive band was determined with Kodak 1D Images analysis software. Optical densities were calculated as arbitrary units after local area background subtraction.

isolated organ studies
The uteri were removed from the 22-day pregnant rats (250-350 g) (n = 8 in each experiment). 5 mm-long muscle rings were sliced from both horns of the uterus and mounted vertically in an organ bath containing 10 mL de Jongh solution (composition: 137 mM NaCl, 3 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 12 mM NaHCO 3 , 4 mM NaH 2 PO 4 , 6 mM glucose, pH = 7.4). The temperature of the organ bath was maintained at 37°C, and carbogen (95% O 2 + 5% CO 2 ) was perfused through the bath. After mounting, the rings were allowed to equilibrate for approximately 60 min be- fore experiments were started, with a buffer change every 15 min. The initial tension of the preparation was set to about 1.5 g and the tension dropped to about 0.5 g by the end of the equilibration period. The tension of the myometrial rings was measured with a gauge transducer (SG-02; Experimetria Ltd, Budapest, Hungary) and recorded with a SPEL Advanced ISOSYS Data Acquisition System (Experimetria Ltd). In the following step contractions were elicited with (-)-noradrenaline (10 −8 to 10 -4.5 M) and cumulative concentration-response curves were constructed in each experiment in the presence of doxazosin (10 −7 M) and propranolol (10 −5 M) in order to avoid α 1 -adrenergic and β-adrenergic actions. Selective α 2 -AR subtype antagonists (each 10 −7 M), propranolol, and doxazosin were left to incubate for 20 minutes before the administration of contracting agents. Following the addition of each concentration of (-)-noradrenaline, recording was performed for 300 s.

Statistical analysis
Concentration-response curves were fitted and areas under curves (AUC) were evaluated and analyzed statistically with the Prism 4.0 (Graphpad Software Inc. San Diego, CA, USA) computer program. From the AUC values, maximum possible effect (E max ) and half maximum effective concentration (EC 50 )values were calculated. ANOVA Dunnett test or two-tailed unpaired t test were used. P < 0.05 was considered as a level of significance. At the end of (-)-noradrenaline incubation period, forskolin (10 −5 M) was added for another 10 min. After stimulation, the samples were immediately frozen in liquid nitrogen and stored until the cAMP extraction (32). Frozen tissue samples were then ground, weighed, homogenized in 10 volumes of ice-cold 5% trichloroacetic acid and centrifuged at 1000g for 10 min. The supernatants were extracted with 3 volumes of water-saturated diethyl ether. After drying, the extracts were stored at -70°C until cAMP assay. Tissue cAMP levels were expressed in pmol/mg tissue.

GTPγS binding assay
The uteri were removed (n = 5 in each experiment) and ho-

RT-PCR and Western blot studies
The mRNA expression of all α 2 -AR subtypes ( Figure 1A, 1C, 1E) was significantly decreased (P < 0.048) after 17βestradiol pretreatment compared to non-treated uteri (P < 0.001). Western blot analysis at the level of protein expression revealed significant decrease (P < 0.027) in each α 2 -AR subtype, corresponding to the PCR results ( Figure  2A-F).
In the presence of the α 2B/C -AR antagonist ARC 239, 17βestradiol pretreatment decreased the myometrial contractions compared to the 17β-estradiol-treated control (P = 0.007) ( Figure 3B) and decreased it (P = 0.045) compared to the ARC 239-treated control ( Table 2).
In the presence of the combination of BRL 44408 and spiroxatrine, 17β-estradiol did not change the maximum myometrial contracting effect of (-)-noradrenaline compared to the 17β-estradiol-treated control (Fig-FiGuRE 1. Changes in the myometrial mRnA expression of the α 2A -(A), α 2B -(B), and α 2C -adrenergic receptors (ARs) (C) after 17β-estradiol pretreatment (n = 5). The statistical analyses were carried out with a two-tailed unpaired t test. **P = 0.005; ***P < 0.001. EC 50 -the concentration of (-)-noradrenaline alone or in the presence of an α 2 -AR antagonist which elicits half of the maximum contracting effect of (-)-noradrenaline. E max -the maximum contracting effect of (-)-noradrenaline alone or in the presence of an α 2 -AR antagonist. ns -not significant. *P < 0.05; **P < 0.01; ***P < 0.001. Significance levels were calculated in comparison with non-treated values.

DiSCuSSion
Since estrogens and the adrenergic system play a major role in myometrial contractions during human gestation, the main focus of our study was to clarify the ef- . and α 2C -ARs (F). The 70, 62, and 60 kDa proteins relate to α 2AR -, α 2B -, and α 2C -ARs and β-actin, respectively. The antibody binding was expressed as optical density (oD) data (A) for α 2AR, (C) for α 2B, and (E) for α 2C -ARs. The y-axis shows the ratio of α 2 -AR/ β-actin protein optical density. The statistical analyses were carried out with a two-tailed unpaired t test. * P < 0.027; *** P < 0.001 fects of estrogen on the α 2 -AR subtypes in late pregnant uterine function in vitro. The estrogen-α 2 -AR connection was investigated via the effects of subtype-selective antagonists after 17β-estradiol pretreatment on the (-)-noradrenaline-stimulated contractions. The experiments were carried out in the presence of the α 1 -AR blocker doxazosin and the β-AR blocker propranolol in order to avoid α 1 -or β-adrenergic actions.
17β-estradiol pretreatment decreased the mRNA and protein expression of the myometrial α 2 -AR subtypes and (-)-noradrenaline-evoked myometrial contraction via the α 2 -ARs, which is similar to our earlier findings with α 1A -ARs (14). According to these findings, we can claim that estrogen differently affects the expression of the α 2 -ARs in various tissues, as it increases the expression of the receptors in the spinal cord and cutaneous arteries (27,28).
In isolated organ bath studies, 17β-estradiol pretreatment decreased (-)-noradrenaline-evoked myometrial contractions via the α 2 -ARs, although it did not modify the myometrial relaxing effect via the α 2A -ARs. However, it abolished the myometrial contraction-increasing effect via the α 2B -ARs. Since there are no available antagonists to produce only α 2C -AR stimulation (ie, α 2A/B -AR blockers), we can only presume that 17β-estradiol did not modify the myometrial relaxing effect via the α 2C -ARs.
To explain why weaker myometrial contractions via the α 2B -AR subtype occurred after 17β-estradiol pretreatment, we measured the myometrial cAMP level, as the changes in the cAMP level are involved in the myometrial effect of the α 2 -ARs. 17β-estradiol pretreatment increased the myometrial cAMP level, which also proves the decreased myometrial contracting effect of (-)-noradrenaline via the α 2 -ARs. It did not modify the cAMP level via the α 2A -ARs, which is in accordance with our previous study (23). However, it increased the myometrial cAMP level via the α 2B -ARs, which can explain the weaker myometrium contracting effect of (-)-noradrenaline.  The α 2 -ARs can couple not only to the G i protein α-subunit, but under certain circumstances, also to G s proteins (24). Estrogen was also shown to decrease the coupling of the α 2 -adrenoceptors to G protein (29). To find an explanation for the cAMP changes, we measured the myometrial [ 35 S] GTPγS binding of the α 2 -AR subtypes after 17β-estradiol pretreatment and in the presence of pertussis toxin, whose inhibitory action is specific for the G i protein.
In the presence of pertussis toxin, 17β-estradiol did not modify the [ 35 S]GTPγS binding of the α 2A -ARs, but it reversed the effect of (-)-noradrenaline on [ 35 S]GTPγS binding via the α 2Aand α 2B -ARs (with spiroxatrine). These findings show that 17β-estradiol modifies the coupling of the α 2B -ARs, but does not change the G protein binding of the α 2A -ARs. To prove this hypothesis, we measured the myometrial [ 35 S] GTPγS binding of the α 2B -AR subtype in the presence of spiroxatrine+BRL 44408. 17β-estradiol decreased the amount of activated G-protein, which is probably a consequence of 17β-estradiol-induced uncoupling of α 2B -ARs from the G proteins (29). This process did not change myometrial contraction as compared with the hormone-treated control.
In the light of our results, we conclude that the functions of the α 2 -AR subtypes are influenced by the female sexual steroid, 17β-estradiol. It decreases the expressions of the α 2 -AR subtypes and increases uterine cAMP level. It does not modify the myometrial relaxing effect via the α 2A -and α 2C -ARs. In case of these receptors we suppose that the 17β-estradiol treatment mainly induces the activation of βγ subunit of G i protein, increasing the smooth muscle cAMP level (17). In case of α 2B -ARs, 17β-estradiol alters the , the α 2C -antagonist spiroxatrine (C), and the BRl 44408-spiroxatrine combination (D) following pretreatment with 17β-estradiol (n = 5). in all cases, the β-adrenoceptors and α 1 -adrenoceptors were inhibited by propranolol and doxazosin. Basal refers to the level of [ 35 S]GTPγS binding without substance. The statistical analyses were carried out with a two-tailed unpaired t test. *P < 0.038; *P < 0.004, ***P < 0.001. myometrial contracting effect of (-)-noradrenaline by reduced coupling of the receptor to G i protein.
A limitation of our study is that we did not carry out any studies on human myometrium, and there might be differences in the function of the rat and human myometrial α 2 -AR subtypes. However, our present findings give a better understanding on the complex physiology of changes during pregnancy, as estrogen is the predominantly expressed hormone during human parturition at term (15,34), which, together with the α 2 -ARs, plays an essential role in myometrial contractility. It was also demonstrated that estrogen level in the amniotic fluid was elevated in uterine inertia (35), which might be caused by the decreased myometrial contractility via the α 2 -AR subtypes. Therefore, estrogen level dysregulation during pregnancy might change the function of the α2-AR subtypes and result in either preterm labor or labor delay. We would like to extend these preclinical studies for premature birth models in rats. We suppose that either subtype-specific agonists or antagonists can be used as a target for drugs against abnormal myometrial contractility.