The effects of progesterone on the alpha2-adrenergic receptor subtypes in late-pregnant uterine contractions in vitro

Background The adrenergic system and progesterone play major roles in the control of the uterine function. Our aims were to clarify the changes in function and expression of the α2-adrenergic receptor (AR) subtypes after progesterone pretreatment in late pregnancy. Methods Sprague Dawley rats from pregnancy day 15 were treated with progesterone for 7 days. The myometrial expressions of the α2-AR subtypes were determined by RT-PCR 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 accumulation of myometrial cAMP was also measured. The activated G-protein level was investigated via GTPγS binding assays. Results Progesterone pretreatment decreased the contractile effect of (−)-noradrenaline through the α2-ARs. The most significant reduction was found through the α2B-ARs. The mRNA of all of the α2-AR subtypes was increased. Progesterone pretreatment increased the myometrial cAMP level in the presence of BRL 44408 (p < 0.001), spiroxatrine (p < 0.001) or the spiroxatrine + BRL 44408 combination (p < 0.05). Progesterone pretreatment increased the G-protein-activating effect of (−)-noradrenaline in the presence of the spiroxatrine + BRL 44408 combination. Conclusions The expression of the α2-AR subtypes is progesterone-sensitive. It decreases the contractile response of (−)-noradrenaline through the α2B-AR subtype, blocks the function of α2A-AR subtype and alters the G protein coupling of these receptors, promoting a Gs-dependent pathway. A combination of α2C-AR agonists and α2B-AR antagonists with progesterone could be considered for the treatment or prevention of preterm birth.


Background
The physiology of uterine quiescence and contractility is very complex. Myometrial contraction is regulated by a number of factors, such as female sexual hormones, the adrenergic receptor (AR) system, ion channels and transmitters. However, the exact cellular and molecular events are still in question. Dysregulation of the myometrial contractility can lead to either preterm or slow-toprogress labor. It is therefore crucial to understand the mechanisms that regulate uterine contractility in order to prevent or treat the pathological processes related to the pregnant myometrium [1][2][3][4].
It is well known that the female sexual hormone progesterone is responsible for uterine quiescence [5,6], while estrogens have major role in myometrial contractions [1,7]. The progesterone level normally declines at term prior to the development of labor and it is therefore used to prevent threatening preterm birth [8,9]. Progesterone and estrogen also play an important role in the regulation of the adrenergic system [10]. Estrogen decreases the expressions of the α 2 -AR subtypes and alters the myometrial contracting effect of (−)-noradrenaline by reduced coupling of the α 2B -ARs to G i protein [11]. Progesterone enhances the synthesis of β 2 -ARs during gestation [12][13][14], and the number of activated G-proteins [12,15], and β 2 -AR agonists can therefore theoretically be combined with progesterone in threatening premature labour [16]. The myometrial α 1 -AR is also influenced by progesterone. It induces a change in the G q /G i -activating property of the α 1AD -AR in rats [17]. However, the effect of progesterone on the myometrial α 2 -AR subtypes is still unknown. Since progesterone has a major role in myometrial quiescence during human parturition [18], it seems important to know its direct influence on the α 2 -AR subtypes, which are also involved in the mechanism of uterine contractions [19].
The α 2 -ARs have been divided into three groups [20,21], the α 2A, α 2B and α 2C subtypes. All of three receptor subtypes bind to the pertussis toxin-sensitive G i protein [22] and decreases the activity of adenylyl cyclase (AC) [23], but under certain circumstances α 2 -ARs can also couple to G s -proteins and increase adenylyl cyclase activity [24]. All three receptor subtypes are involved in various physiological functions, and especially in the cardiovascular and central nervous systems [25]. Furthermore, all of them have been identified in both the pregnant and the non-pregnant myometrium, and have been shown to take part in both increased and decreased myometrial contractions [26]. The α 2B -ARs predominate and mediate contraction at the end of gestation in rats, decreasing the intracellular cAMP level, while the stimulation of the myometrial α 2A -and α 2C -ARs increases the cAMP level, and mediates only weak contractions [27].
Since no data are available on the effects of progesterone on the myometrial functions of the different α 2 -AR subtypes, we set out to clarify the changes in expression and function of the α 2A -, α 2B -and α 2C -AR subtypes after progesterone pretreatment on the last day of pregnancy in rats.

Methods
The animal experimentation was carried out with the approval of the Hungarian Ethical Committee for Animal Research (permission number: IV/198/2013). The animals were treated in accordance with the EU Directive 2010/63/EU for animal experiments 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 kept on a standard rodent pellet diet (INNOVO Ltd., Isaszeg, Hungary), with tap water available ad libitum.

Mating of the animals
Mature female (180-200 g) and male (240-260 g) Sprague-Dawley rats were mated in a special mating cage in the early morning hours. A time-controlled metal door separated the rooms for the male and female animals. The separating door was opened before dawn (4 a.m.) Within 4-5 h after the possibility of mating, intercourse was confirmed by the presence of a copulation plug or vaginal smears. In positive cases, the female rats were separated and this was regarded as the first day of pregnancy.

In vivo sexual hormone treatments of the rats
The progesterone (Sigma-Aldrich, Budapest, Hungary) pretreatment of the pregnant animals was started on day 15 of pregnancy. Progesterone was dissolved in olive oil and injected subcutaneously every day up to day 21 in a dose of 0.5 mg/0.1 ml [28].
On day 22, the uterine samples were collected, and contractility and molecular pharmacological studies were carried out.

RT-PCR studies
Tissue isolation: Rats (250-350 g) were sacrificed by CO 2 asphyxiation. Fetuses were sacrificed by immediate cervical dislocation. The uterine tissues from pregnant animals (tissue between two implantation sites) were rapidly removed and placed in RNAlater Solution (Sigma-Aldrich, Budapest, Hungary). The tissues were frozen in liquid nitrogen and then stored at −70°C until the extraction of total RNA. Total RNA preparation from tissue: Total cellular RNA was isolated by extraction with guanidinium thiocyanate-acid-phenol-chloroform according to the procedure of Chomczynski and Sacchi [29]. 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 s and 60°C for 1 min. The generation of specific PCR products was confirmed by melting curve analysis. Table 1 contains 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
Twenty μg of protein per well was subjected to electrophoresis on 4-12% NuPAGE Bis-Tris Gel in XCell Sure-Lock Mini-Cell Units (Life Technologies, Budapest, Hungary). Proteins were transferred from gels to nitrocellulose membranes, using the iBlot Gel Transfer System (Life Technologies, Hungary). The antibody binding was detected with the WesternBreeze Chromogenic Western blot immundetection kit (Life Technologies, Budapest, Hungary). 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, California, 1:200) in the blocking buffer. Images were captured with the EDAS290 imaging system (Csertex Ltd., 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
Uteri were removed from the 22-day-pregnant rats (250-350 g). 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 before 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., Budapest, Hungary). 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 1adrenergic and β-adrenergic actions. Selective α 2 -AR subtype antagonists (each 10 −7 M), propranolol and doxazosin were left to incubate for 20 min before the administration of contracting agents. Following the addition of each concentration of (−)-noradrenaline, recording was performed for 300 s. Concentration-response curves were fitted and areas under curves (AUC) were evaluated and analysed statistically with the Prism 4.0 (Graphpad Software Inc. San Diego, CA, USA) computer program. From the AUC values, E max and EC 50 values were calculated. Statistical evaluations were carried out with the ANOVA Dunnett test or the two-tailed unpaired t-test.

Measurement of uterine cAMP accumulation
Uterine cAMP accumulation was measured with a commercial cAMP Enzyme Immunoassay Kit (Cayman Chemical, USA). Uterine tissue samples (control and 17β-estradiol-treated) from 22-day-pregnant rats were incubated in an organ bath (10 ml) containing de Jongh solution (37°C, perfused with carbogen). Isobutylmethylxanthine (10 −3 M), doxazosin (10 −7 M), propranolol (10 −5 M) and the investigated subtype-selective α 2 -AR antagonists (each 10 −7 M) were incubated with the tissues for 20 min, and (−)-noradrenaline (3 × 10 −6 M) were added to the bath for 10 min. At the end of the (−)-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 extraction of cAMP [30]. 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 the cAMP assay. Uterine cAMP accumulation was measured with a commercial competitive cAMP EIA Kit; tissue cAMP levels were expressed in pmol/mg tissue.

RT-PCR and Western blot studies
The mRNA expression of each α 2 -AR subtype (Fig. 1a-c) was significantly increased after progesterone pretreatment as compared with the non-treated uteri (p < 0.05). The results of Western blot analysis at the level of protein expression revealed a significant increase in each α 2 -AR subtype, which correlated with the PCR results ( Fig. 2a-f ).
In the presence of the α 2B/C -AR antagonist ARC 239, progesterone pretreatment did not modify the myometrial contracting effect of (−)-noradrenaline relative to the progesterone-treated control. The concentrationresponse curve was very flat, the difference between the Fig. 1 Changes in the myometrial mRNA and protein expression of the α 2A -(a), α 2B -(b) and α 2C -adrenergic receptors (c) after progesterone pretreatment. The statistical analyses were carried out with the two-tailed unpaired t-test. * p < 0.05 ; ** p < 0.01 minimum and the maximum effect was less then 20% (Fig. 3b). ARC 239 reduced the (−)-noradrenaline-induced contractions, which were decreased further by progesterone pretreatment (p < 0.05) (Fig. 3a, b; Table 2c).
In the presence of the combination of spiroxatrine + BRL 44408, progesterone pretreatment did not modify the maximum myometrial contracting effect of (−)-noradrenaline in comparison with the progesterone-treated control (Fig. 3b). The combination of the two compounds increased the (−)-noradrenaline-induced contractions, which were reduced by progesterone pretreatment (p < 0.001) (Fig. 3a, b; Table 2e).

cAMP studies
Progesterone pretreatment increased the myometrial cAMP level (p < 0.05) (Fig. 4) produced in the presence of (−)-noradrenaline. The myometrial cAMP level was also increased in the presence of BRL 44408 (p < 0.001), spiroxatrine (p < 0.001) and the spiroxatrine + BRL 44408 combination (p < 0.05). However, ARC 239 did not modify the amount of myometrial cAMP level after progesterone pretreatment. In addition, BRL 44408 (p < 0.05) and spiroxatrine (p < 0.01) increased the myometrial cAMP level compared to the progesterone-treated control.
In the presence of ARC 239, (−)-noradrenaline moderately increased the [ 35 S]GTPγS binding and it was more elevated after progesterone pretreatment (p < 0.01). In the presence of pertussis toxin, the [ 35 S]GTPγS bindingstimulating effect of (−)-noradrenaline ceased, which was not modified even by progesterone pretreatment (Fig. 5b).
In the presence of spiroxatrine, (−)-noradrenaline slightly increased the [ 35 S]GTPγS binding and it was more elevated (p < 0.001) after progesterone pretreatment. In the presence of pertussis toxin, however,  (Fig. 5c).
In the presence of the spiroxatrine + BRL 44408 combination, (−)-noradrenaline inhibited the [ 35 S]GTPγS binding, but it was significantly increased after progesterone pretreatment (p < 0.001). In the presence of pertussis toxin, the spiroxatrine + BRL 44408 combination caused a dose-dependent inhibition in the [ 35 S]GTPγS binding of (−)-noradrenaline, but the inhibition was reduced after progesterone pretreatment (Fig. 5d).

Discussion
Since progesterone and the adrenergic system play major roles in the myometrial function during gestation, the main focus of our study was to clarify the effects of progesterone on the α 2 -AR subtypes in the late-pregnant uterine function in vitro. The α 2 -AR-selective action of (−)-noradrenaline was provided by the application of the α 1 -blocker doxazosin and the β-AR blocker propanolol. The applications of subtype-selective antagonists gave us the possibility to investigate the subtype-specific α 2 -AR responses to (−)-noradrenaline and to detect the modification induced by progesterone pretreatment. In an earlier study, we determined the subtype-selective α 2 -AR action of (−)-noradrenaline, and our present work therefore focused mainly on the influence of progesterone as a modifier of the α 2 -AR response [27].
Progesterone pretreatment increased the mRNA and protein expression of the myometrial α 2 -AR subtypes, but decreased the (−)-noradrenaline-evoked myometrial contraction through the α 2 -ARs, which was similar to our earlier findings with the α 1 -ARs [17]. , the α 2C -adrenergic receptor antagonist spiroxatrine (c) and the spiroxatrine + BRL 44408 combination (d) following pretreatment with progesterone. In all cases, the β-adrenergic receptors and the α 1 -adrenergic receptors 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 the ANOVA Dunnett test. **p < 0.01; ***p < 0.001 In the isolated organ bath studies, progesterone pretreatment ceased the (−)-noradrenaline-evoked myometrial contraction through the α 2 -ARs, although it practically ceased the myometrial contracting effect of the (−)-noradrenaline through the α 2A -ARs. Additionally, it abolished the myometrial contraction-increasing effect through the α 2B -ARs, and reversed the myometrial contracting effect in the presence of BRL 44408 and in the presence of spiroxatrine. Since there are no available α 2A/B -AR blockers to produce only α 2C -AR stimulation, we can only presume that progesterone maintained the myometrial relaxing effect through the increased number and function of α 2C -ARs.
To find an explanation of the weaker myometrial contractions via the α 2B -AR subtype after progesterone pretreatment, we measured the myometrial cAMP level, since the changes in the cAMP level are involved in the myometrial effect of the α 2 -ARs. Progesterone pretreatment increased the myometrial cAMP level, which additionally proves the decreased myometrial contracting effect of (−)-noradrenaline through the α 2 -ARs. It did not alter the cAMP level through the α 2A -ARs, which is in harmony with the result of the isolated organ bath studies that (−)-noradrenaline did not influence the myometrial contractions via these receptors after progesterone pretreatment. However, it increased the myometrial cAMP level through the α 2B -ARs, which can explain the weaker myometrium-contracting effect of (−)-noradrenaline in the presence of BRL 44408 (stimulation via α 2B -and α 2C -ARs), spiroxatrine (stimulation via α 2A -and α 2B -ARs) and the spiroxatrine + BRL 44408 combination (stimulation via α 2B -AR).
The literature indicates that the G i /G s -activating property of α 2 -AR in rats changes during gestation, resulting in differences in the regulation of myometrial adenylyl cyclase activity at mid-pregnancy versus term [32]. Moreover, progesterone induces a change in the G q /G iactivating property of α 1AD -AR in rats [17]. We therefore measured whether progesterone can modify the myometrial [ 35 S]GTPγS binding of the α 2 -AR subtypes in the presence of the G i protein blocker pertussis toxin at the end of pregnancy. Progesterone did not modify the [ 35 S]GTPγS binding of the α 2A -ARs. However, via the α 2A -and α 2B -ARs (with spiroxatrine), progesterone reversed the effect of (−)-noradrenaline on the [ 35 S]GTPγS binding in the presence of pertussis toxin and also increased the [ 35 S]GTPγS binding-stimulating effect of (−)-noradrenaline. These findings indicate that progesterone modifies the coupling of α 2B -ARs, but not the G protein binding of the α 2A -ARs. To confirm this hypothesis, we measured the myometrial [ 35 S]GTPγS binding of the α 2B -AR subtype in the presence of the spiroxatrine + BRL 44408 combination. Progesterone reversed the effect of (−)-noradrenaline on [ 35 S]GTPγS binding in the presence of pertussis toxin and also reversed the [ 35 S]GTPγS binding-stimulating effect of (−)-noradrenaline. This result suggests that, in the presence of predominance of progesterone, the α 2B -ARs are coupled, at least partially, to G s protein, which leads to the activation of adenylyl cyclase and decreases the (−)-noradrenaline-induced myometrial contraction via these receptors.

Conclusions
We conclude that progesterone increases the expression of each α 2 -AR subtype, and reduces the (−)-noradrenalineinduced myometrial contractions via the totality of these receptors. Progesterone blocks the G-protein coupling and cAMP production via the α 2A -ARs. In the case of the α 2C -ARs, we presume that progesterone treatment mainly induces the activation of the βγ subunit of the G i protein, eliciting an increase in the smooth muscle cAMP level [19]. In the case of the α 2B -ARs, G s coupling is a determining factor in the function of the receptors after progesterone treatment, which leads to an increased cAMP level and decreased myometrial contraction.
Since the myometrial sensitivity to progesterone decreases at term, we assume that these changes can lead to the increased myometrial contraction near term via the α 2 -ARs. We presume that the effects of α 2C -AR agonists and α 2B -AR antagonists in combination with progesterone may open up new targets for drugs against premature birth.