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The Effects of Progesterone on the Alpha2-Adrenergic Receptor

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The Effects of Progesterone on the Alpha2-Adrenergic Receptor Hajagos-Tóth et al. Reproductive Biology and Endocrinology (2016) 14:33 DOI 10.1186/s12958-016-0166-9 RESEARCH Open Access The effects of progesterone on the alpha2-adrenergic receptor subtypes in late-pregnant uterine contractions in vitro Judit Hajagos-Tóth1, Judit Bóta1, Eszter Ducza1, Reza Samavati2, Anna Borsodi2, Sándor Benyhe2 and Róbert Gáspár1* Abstract 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. Keywords: Progesterone, Gestation, Rat, Myometrium, α2-adrenergic receptor subtypes Background to prevent or treat the pathological processes related to The physiology of uterine quiescence and contractility is the pregnant myometrium [1–4]. very complex. Myometrial contraction is regulated by a It is well known that the female sexual hormone pro- number of factors, such as female sexual hormones, the gesterone is responsible for uterine quiescence [5, 6], adrenergic receptor (AR) system, ion channels and while estrogens have major role in myometrial contrac- transmitters. However, the exact cellular and molecular tions [1, 7]. The progesterone level normally declines at events are still in question. Dysregulation of the myome- term prior to the development of labor and it is there- trial contractility can lead to either preterm or slow-to- fore used to prevent threatening preterm birth [8, 9]. progress labor. It is therefore crucial to understand the Progesterone and estrogen also play an important role in mechanisms that regulate uterine contractility in order the regulation of the adrenergic system [10]. Estrogen decreases the expressions of the α2-AR subtypes and al- ters the myometrial contracting effect of (−)-noradren- * Correspondence: [email protected] 1Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy, aline by reduced coupling of the α2B-ARs to Gi protein University of Szeged, Szeged H-6701, P.O. Box 121, Hungary [11]. Progesterone enhances the synthesis of β2-ARs Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hajagos-Tóth et al. Reproductive Biology and Endocrinology (2016) 14:33 Page 2 of 9 during gestation [12–14], and the number of activated Mating of the animals G-proteins [12, 15], and β2-AR agonists can therefore Mature female (180–200 g) and male (240–260 g) theoretically be combined with progesterone in threaten- Sprague–Dawley rats were mated in a special mating ing premature labour [16]. The myometrial α1-AR is also cage in the early morning hours. A time-controlled influenced by progesterone. It induces a change in the metal door separated the rooms for the male and female Gq/Gi-activating property of the α1AD-AR in rats [17]. animals. The separating door was opened before dawn However, the effect of progesterone on the myometrial (4 a.m.) Within 4–5 h after the possibility of mating, α2-AR subtypes is still unknown. Since progesterone has intercourse was confirmed by the presence of a copula- a major role in myometrial quiescence during human tion plug or vaginal smears. In positive cases, the female parturition [18], it seems important to know its direct rats were separated and this was regarded as the first influence on the α2-AR subtypes, which are also involved day of pregnancy. in the mechanism of uterine contractions [19]. The α2-ARs have been divided into three groups [20, In vivo sexual hormone treatments of the rats 21], the α2A, α2B and α2C subtypes. All of three receptor The progesterone (Sigma-Aldrich, Budapest, Hungary) subtypes bind to the pertussis toxin-sensitive Gi protein pretreatment of the pregnant animals was started on day [22] and decreases the activity of adenylyl cyclase (AC) 15 of pregnancy. Progesterone was dissolved in olive oil [23], but under certain circumstances α2-ARs can also and injected subcutaneously every day up to day 21 in a couple to Gs-proteins and increase adenylyl cyclase ac- dose of 0.5 mg/0.1 ml [28]. tivity [24]. All three receptor subtypes are involved in On day 22, the uterine samples were collected, and various physiological functions, and especially in the car- contractility and molecular pharmacological studies were diovascular and central nervous systems [25]. Further- carried out. more, all of them have been identified in both the pregnant and the non-pregnant myometrium, and have RT-PCR studies been shown to take part in both increased and decreased Tissue isolation: Rats (250–350 g) were sacrificed by myometrial contractions [26]. The α2B-ARs predominate CO2 asphyxiation. Fetuses were sacrificed by immediate and mediate contraction at the end of gestation in rats, cervical dislocation. The uterine tissues from pregnant decreasing the intracellular cAMP level, while the stimu- animals (tissue between two implantation sites) were lation of the myometrial α2A- and α2C-ARs increases the rapidly removed and placed in RNAlater Solution cAMP level, and mediates only weak contractions [27]. (Sigma-Aldrich, Budapest, Hungary). The tissues were Since no data are available on the effects of progester- frozen in liquid nitrogen and then stored at −70 °C until one on the myometrial functions of the different α2-AR the extraction of total RNA. subtypes, we set out to clarify the changes in expression Total RNA preparation from tissue: Total cellular and function of the α2A-, α2B- and α2C-AR subtypes after RNA was isolated by extraction with guanidinium progesterone pretreatment on the last day of pregnancy thiocyanate-acid-phenol-chloroform according to the in rats. procedure of Chomczynski and Sacchi [29]. After pre- cipitation with isopropanol, the RNA was washed with 75% ethanol and then re-suspended in diethyl Methods pyrocarbonate-treated water. RNA purity was controlled The animal experimentation was carried out with the at an optical density of 260/280 nm with BioSpec Nano approval of the Hungarian Ethical Committee for (Shimadzu, Japan); all samples exhibited an absorbance Animal Research (permission number: IV/198/2013). ratio in the range 1.6–2.0. RNA quality and integrity The animals were treated in accordance with the EU were assessed by agarose gel electrophoresis. Directive 2010/63/EU for animal experiments and the Reverse transcription and amplification of the PCR Hungarian Act for the Protection of Animals in Research products was performed by using the TaqMan RNA-to- (XXVIII. tv. 32.§). CTTM 1-Step Kit (Life Technologies, Budapest, Hungary) and the ABI StepOne Real-Time cycler. RT-PCR amplifi- Housing and handling of the animals cations were performed as follows: 48 °C for 15 min and Sprague–Dawley rats were obtained from the INNOVO 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s Ltd. (Gödöllő, Hungary) and were housed under con- and 60 °C for 1 min. The generation of specific PCR prod- trolled temperature (20–23 °C), in humidity (40–60%) ucts was confirmed by melting curve analysis. Table 1 and light (12 h light/dark regime)-regulated rooms. The contains the assay IDs for the used primers. The amplifi- animals were kept on a standard rodent pellet diet cation of β-actin served as an internal control. All samples (INNOVO Ltd., Isaszeg, Hungary), with tap water avail- were run in triplicates. The fluorescence intensities of the able ad libitum. probes were plotted against PCR cycle numbers. The Hajagos-Tóth et al. Reproductive Biology and Endocrinology (2016) 14:33 Page 3 of 9 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) TaqMan assays Assay ID (Life Technologies, Hungary) Accession number Assay location Amplicon length Annealing temp.
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