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-to-progress 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‐
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‐
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 α2-AR subtypes, we set out to clarify the changes in expression and function of the α2A-, α2B- and α2C-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 CO2 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).
Table 1
Parameters of the applied primers and PCR reactions. The real-time reverse transcription polymerase chain reactions were used to determine the changes in the mRNA expression. In our studies the parameters of inventoried TaqMan assays were defined by Life Technologies (ThermoFisher Scientific, Hungary)
α2A-AR | Rn00562488_s1 | NM_012739.3 | 1350 | 72 | 60 | 20 |
α2B-AR | Rn00593312_s1 | NM_138505.2 | 1451 | 63 | 60 | 20 |
α2C-AR | Rn00593341_s1 | NM_138506.1 | 653 | 111 | 60 | 20 |
β-actin | Rn00667869_m1 | NM_031144.3 | 881 | 91 | 60 | 20 |
Western blot analysis
Twenty μg of protein per well was subjected to electrophoresis on 4–12% NuPAGE Bis-Tris Gel in XCell SureLock 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 α2A-AR, α2B-AR, α2C-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 CaCl2, 1 mM MgCl2, 12 mM NaHCO3, 4 mM NaH2PO4, 6 mM glucose, pH = 7.4). The temperature of the organ bath was maintained at 37 °C, and carbogen (95% O2 + 5% CO2) 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 α1-adrenergic and β-adrenergic actions. Selective α2-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, Emax and EC50 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 1000
g 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.
[35S]GTPγS binding assay
Uteri were removed and homogenized in 20 volumes (w/v) of ice-cold buffer (10 mM Tris–HCl, 1 mM EDTA, 0.6 mM MgCl
2, and 0.25 M sucrose, pH 7.4) with an Ultra Turret T25 (Janke & Kunkel, Staufen, Germany) homogenizer, and the suspension was then filtered on four layers of gauze and centrifuged (40,000
g, 4 °C, 20 min). After centrifugation, the pellet was resuspended in a 5-fold volume of buffer. The protein contents of the samples were diluted to 10 mg protein/sample. Membrane fractions was incubated in a final volume of 1 ml at 30 °C for 60 min in Tris-EGTA buffer (pH 7.4) composed of 50 mM Tris–HCl, 1 mM EGTA, 3 mM MgCl
2, 100 mM NaCl, containing 20 MBq/0.05 cm
3 [
35S]GTPγS (0.05 nM) (Sigma Aldrich, Budapest, Hungary), together with increasing concentrations (10
−9–10
−5 M) of (−)-noradrenaline. BRL 44408, ARC 239 and spiroxatrine were used in a fixed concentration of 0.1 μM. For the blocking of α
1- and β-ARs, doxazosin and propranolol were used in a fixed concentration of 10 μM. Total binding was measured in the absence of the ligands, non-specific binding was determined in the presence of 10 μM unlabeled GTPγS and subtracted from total binding. The difference represents basal activity. Bound and free [
35S]GTPγS were separated by vacuum filtration through Whatman GF/B filters with Brandel M24R Cell harvester. Filters were washed three times with 5 ml ice-cold buffer (pH 7.4), and the radioactivity of the dried filters was detected in UltimaGold™ MV scintillation cocktail with Packard Tricarb 2300TR liquid scintillation counter [
31]. The [
35S]GTPγS binding experiments were performed in triplicate and repeated at least three times.
Gi protein was inhibited with pertussis toxin (Sigma Aldrich, Budapest, Hungary) in a concentration of 500 ng/ml after the addition of protein and GDP to the Tris-EGTA buffer 30 min before [35S]GTPγS.
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].
In the isolated organ bath studies, progesterone pretreatment ceased the (−)-noradrenaline-evoked myometrial contraction through the α2-ARs, although it practically ceased the myometrial contracting effect of the (−)-noradrenaline through the α2A-ARs. Additionally, it abolished the myometrial contraction-increasing effect through the α2B-ARs, and reversed the myometrial contracting effect in the presence of BRL 44408 and in the presence of spiroxatrine. Since there are no available α2A/B-AR blockers to produce only α2C-AR stimulation, we can only presume that progesterone maintained the myometrial relaxing effect through the increased number and function of α2C-ARs.
To find an explanation of the weaker myometrial contractions via the α2B-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 α2-ARs. Progesterone pretreatment increased the myometrial cAMP level, which additionally proves the decreased myometrial contracting effect of (−)-noradrenaline through the α2-ARs. It did not alter the cAMP level through the α2A-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 α2B-ARs, which can explain the weaker myometrium-contracting effect of (−)-noradrenaline in the presence of BRL 44408 (stimulation via α2B- and α2C-ARs), spiroxatrine (stimulation via α2A- and α2B-ARs) and the spiroxatrine + BRL 44408 combination (stimulation via α2B-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
i-activating property of α
1AD-AR in rats [
17]. We therefore measured whether progesterone can modify the myometrial [
35S]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 [
35S]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 [
35S]GTPγS binding in the presence of pertussis toxin and also increased the [
35S]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 [
35S]GTPγS binding of the α
2B-AR subtype in the presence of the spiroxatrine + BRL 44408 combination. Progesterone reversed the effect of (−)-noradrenaline on [
35S]GTPγS binding in the presence of pertussis toxin and also reversed the [
35S]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 (−)-noradrenaline-induced 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 α2-ARs. We presume that the effects of α2C-AR agonists and α2B-AR antagonists in combination with progesterone may open up new targets for drugs against premature birth.
Abbreviations
AR, Adrenergic receptor; cAMP, Cyclic adenosine monophosphate; EC50, Half of the maximum effect; Emax, Maximum effect; G protein, Heterotrimeric guanine nucleotide binding regulatory protein; GTPγS, Guanosine-5′-O-(γ-thio)triphosphate; NA, Noradrenaline; PTX, Pertussis toxin; RT-PCR, Reverse transcriptase-polymerase chain reaction; s.c., Subcutaneous; Tris–HCl, Tris(hidroxymethyl)aminomethane
Funding
The study was supported by a grant from the Hungarian National Research, Development and Innovation Office (NKFI), Budapest, Hungary; OTKA-108518.