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The Effects of Estrogen on the Α2-Adrenergic Receptor Subtypes In

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The Effects of Estrogen on the Α2-Adrenergic Receptor Subtypes In 100 RECOOP for Common Mechanisms of Diseases Croat Med J. 2016;57:100-9 doi: 10.3325/cmj.2016.57.100 The effects of estrogen on Judit Hajagos-Tóth1, Judit Bóta1, Eszter Ducza1, the α2-adrenergic receptor Adrienn Csányi1, Zita Tiszai1, Anna Borsodi2, Reza subtypes in rat uterine function Samavati2, Sándor Benyhe2, in late pregnancy in vitro Róbert Gáspár1 1Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy, University of Szeged, Szeged, Hungary 2Institute of Biochemistry, Biological Aim To assess the effect of 17β-estradiol pretreatment on Research Centre, Hungarian Academy of Sciences, Szeged, the function and expression of α2- adrenergic receptors (ARs) subtypes in late pregnancy in rats. Hungary Methods Sprague-Dawley rats (n = 37) were treated with 17β-estradiol for 4 days starting from the 18th day of preg- nancy. The myometrial expression of the α2-AR subtypes was determined by real time polymerase chain reaction and Western blot analysis. In vitro contractions were stim- ulated with (-)-noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (α2A), ARC 239 (α2B/C), and spiroxatrine (α2A). The cyclic adenosine mono- phosphate (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 con- tractile effect of (-)-noradrenaline via the 2α -ARs, and abol- ished the contractile effect via the 2Bα -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 sen- sitive to 17β-estradiol, which decreases the contractile re- sponse of (-)-noradrenaline via the α2B-AR subtype, and might cause changes in G-protein signaling pathway. Es- trogen dysregulation may be responsible for preterm labor Received: January 8, 2016 or uterine inertia via the α -ARs. 2 Accepted: March 22, 2016 Correspondence to: Róbert Gáspár Szeged, H-6701 P.O. Box 121, Hungary [email protected] www.cmj.hr Hajagos-Tóth et al: Effect of estrogen on the myometrial α2-adrenergic receptor subtypes 101 In spite of the numerous attempts to explore it, the exact increasing the cAMP level, which can be regarded as re- action mechanism and risk of preterm birth still remains laxation as they are compared with the effect of (-)-nora- one of the biggest challenges in obstetrics and gynecolo- drenaline (23). gy and a major contributor to perinatal mortality and mor- bidity, affecting around 9% of births in developed coun- Since female sexual steroid hormones play an important tries (1-4). On the other hand, weak contractions and poor role in the regulation of the adrenergic receptor system labor outcomes also represent a problem mainly among (26), the effect of estrogen on different α2-AR subtypes has obese women, increasing the number of cesarean deliv- been investigated. The mRNA expression of the α2A-ARs in eries (5). the spinal cord was increased after estrogen pretreatment (27), which could contribute to the higher prevalence of Uterine contractility is regulated by several factors, such pain syndromes in women. On the other hand, estrogen as the adrenergic system (6) and female sexual hormones was shown to increase the smooth muscle expression of (7,8). Progesterone was demonstrated to increase the syn- α2C-ARs and therefore the cold-induced constriction of cu- thesis of β2-ARs during pregnancy (9-11) and the num- taneous arteries (28). In addition, it was shown to stimu- ber of activated G-proteins (12,13), which is why it can be late the (-)-noradrenaline release in the hypothalamus due combined with β2-AR agonists in threatening preterm la- to the decreased coupling of the α2-adrenoceptors to G bor. Myometrial α1-AR expression is influenced by female protein (29). sexual steroid hormones, mainly estrogens. 17β-estradiol decreases the expression of the α1A-ARs, but does not influ- Since there are no available data on the effects of 17β- ence the expression of α1D-ARs (14). However, the effect of estradiol on the myometrial functions of different α2-AR estrogens on the myometrial α2-AR subtypes is unknown. subtypes, the aim of this study was to clarify the changes Considering the fact that estrogens play a major role in my- in expression and function of the α2A-, α2B-, and α2C-AR sub- ometrial contractions during human parturition (15,16), it types after 17β-estradiol pretreatment on the last day of is important to know if they have a direct influence on the pregnancy in rats by using RT-PCR and Western blot analy- α2-AR subtypes, which are also involved in the mechanism sis. Since the changes in the intracellular cAMP are crucial of uterine contractions (17). in the control of smooth muscle contractions and relax- ations, our further aim was to measure the cAMP release The α2-ARs have been divided into (18,19) α2A, α2B, and α2C after 17β-estradiol pretreatment in the presence of the subtypes. All three receptor subtypes are coupled to the subtype-specific 2α -AR antagonists. We also investigated pertussis toxin-sensitive Gi-protein α-subunit (20) and de- the changes in the G-protein activation of α2-ARs using crease the activity of adenylyl cyclase (AC) and voltage- GTPγS binding assay. gated Ca2+ currents, at the same time activating the re- ceptor-operated K+ currents (21). The stimulation of these MaterIALS AND methODS receptors leads to presynaptic feedback inhibition of (-)-noradrenaline release on the adrenergic neurons (18), The animal experimentation was carried out with the ap- and mediates a variety of cell functions, such as vaso- proval of the Hungarian Ethics Committee for Animal Re- constriction, increased blood pressure, and nociception. search (permission number: IV/198/2013). The animals Furthermore, all three α2-AR subtypes were identified in were treated in accordance with the European Commu- both pregnant and non-pregnant myometrium and were nities Council Directives (86/609/ECC) and the Hungar- shown to take part in both increased and decreased myo- ian Act for the Protection of Animals in Research (XXVIII. metrial contractions (22,23). Under certain circumstances, tv. 32.§). α2-ARs can couple not only to Gi-proteins but to Gs-pro- teins, resulting in the activation of AC (24). On the other Housing and handling of the animals hand, pregnancy has been proved to induce a change in the Gi/Gs-activating property of the α2-ARs in rats, result- Sprague-Dawley rats were obtained from the INNOVO Ltd ing in a differential regulation of myometrial AC activity (Gödöllő, Hungary) and were housed under controlled in mid-pregnancy vs term (25). The α2B-ARs were shown temperature (20-23°C), in humidity (40%-60%) and light to predominate and mediate contraction in last-day-preg- (12 h light/dark regime) regulated rooms. The animals nant animals by decreasing the intracellular cAMP level, were fed standard rodent pellet diet (INNOVO Ltd, Isaszeg, while α2A- and α2C-ARs mediate only weak contractions by Hungary), with tap water available ad libitum. www.cmj.hr 102 RECOOP for Common Mechanisms of Diseases Croat Med J. 2016;57:100-9 Mating of the animals 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. Mature female (180-200 g, n = 58) and male (240-260 g, The generation of specific PCR products was confirmed by n = 12) Sprague-Dawley rats were mated in a special mat- melting curve analysis. Table 1 shows the assay IDs for the ing cage with a time-controlled electrically movable metal used primers. The amplification of β-actin served as an in- door separating the rooms for male and female animals. ternal control. All samples were run in triplicates. The flu- Since rats are usually active at night, the door was opened orescence intensities of the probes were plotted against before dawn. Within 4-5 hours after the possibility of mat- PCR cycle numbers. The amplification cycle displaying the ing, female rats with the presence of copulation plug or a first significant increase in the fluorescence signal was de- sperm-positive vaginal smear (search was performed un- fined as the threshold cycle (Ct). der under a microscope at a magnification of 1200 times) TabLE 1. Assay IDs of the applied primers were separated. The day of copulation was considered as the first day of pregnancy. Assay ID TaqMan assays (Life Technologies, Budapest, Hungary) α -AR Rn00562488_s1 In vivo sexual hormone treatments of the rats 2A α2B-AR Rn00593312_s1 α2C-AR Rn00593341_s1 The 17β-estradiol (Sigma Aldrich, Budapest, Hungary) pre- β-actin Rn00667869_m1 treatment 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 Western blot analysis 17β-estradiol once a day for 4 days (30). On the day 22, the uterine samples were collected and the contractility and 20 μg of protein per well was subjected to electrophoresis molecular pharmacological studies were carried out. on 4%-12% NuPAGE Bis-Tris Gel in XCell SureLock Mini-Cell Units (Life Technologies) (n = 5 for each α2-AR subtype an- RT-PCR studies tagonists). Proteins were transferred from gels to nitrocel- lulose membranes, using the iBlot Gel Transfer System (Life Tissue isolation: Rats (250-300 g) were sacrificed by CO2 Technologies).
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