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Journal of Animal Science - Animal Physiology

β2– and β3–adrenergic receptors stimulation relaxes porcine myometrium in the peri-implantation period12

 

This article in JAS

  1. Vol. 94 No. 11, p. 4611-4618
     
    Received: Apr 26, 2016
    Accepted: Aug 28, 2016
    Published: October 27, 2016


    3 Corresponding author(s): mark@uwm.edu.pl
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doi:10.2527/jas.2016-0577
  1. W. Markiewicz 3a and
  2. J. J. Jaroszewskia
  1. a Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, 10-718 Olsztyn, Poland

Abstract

These studies were to demonstrate whether the β3–receptor is involved in regulating the contractility of the swine myometrium in the peri-implantation period and to compare a relaxing activity of selective β2– and β3–adrenergic agonists (salbutamol and BRL 37344, respectively). Myometrial strips were collected form the gilts (n = 8) on d 12 to 14 of pregnancy. Changes in the contractility of myometrial strips were assessed further to the administration of increasing concentrations of the agonists (10–9 to 10–5 M) with and without β-adrenergic receptor antagonists: butoxamine, propranolol, and bupranolol at 10–4 M. Moreover, the –log EC50 (pD2) of the agonists were compared. Following the incubation of myometrial strips with salbutamol and BRL 37344, a significant (P < 0.05 to 0.001) reduction in the contractility, seen as decreased tension, amplitude, and frequency of contractions, as compared to the pretreatment period was noted, although salbutamol induced these changes at a concentration of 10–8 to 10–5 M, whereas BRL 37344 induced them at 10–7 to 10–5 M. Salbutamol exerted a significantly (P < 0.001) more potent reduction of the tension (pD2 = 6.98 ± 0.93 [SD]) than BRL 37344 (pD2 = 3.41 ± 0.1.61). The impact of salbutamol was neutralized by all administered antagonists, while a relaxing effect of BRL 37344 was completely inhibited by bupranolol and partially by propranolol. The data indicate that the β3–adenergic receptor is involved in the regulation of smooth muscle contractility in the swine uterus in the peri-implantation period and its activation triggers relaxation of the myometrium to a lesser degree than the activation of the β2–adrenergic receptor.



INTRODUCTION

It was initially believed that β2–adrenoceptors play a predominant role in the relaxation of rat (Engstrom et al., 1997) and human (Liu et al., 1998) myometrium. However, the presence of functional β3–adrenoceptors has been demonstrated in human near-term (Bardou et al., 2000) and human nonpregnant myometrium (Rouget et al., 2005) as well as in pregnant rat (Minorics et al., 2009) and pregnant mouse (Parida et al., 2013) myometrium. Currently, a predominant role of the β3–adrenoceptor in human myometrium contractility is suspected (Rouget et al., 2005; Ursino et al., 2009).

In gilts, myometrial activity undergoes changes during the oestrous cycle (Kitazawa et al., 2001; Cao et al., 2002; Kucharski et al., 2007; Jana et al., 2010; Jana et al., 2013) and during the course of pregnancy (Kitazawa et al., 2003; Kurowicka et al., 2005; Markiewicz et al., 2016). The early pregnancy in the pig is divided into 3 periods: postconception (d 1 to 10 of pregnancy), the maternal recognition of pregnancy (d 11 to 13), and implantation (d 14 to 19; Zięcik et al., 2011). Thus, the period between d 12 and 14 of the pregnancy is crucial for successful implantation, and the motor activity of the uterus at this time is particularly important for the migration of embryos. The effect of various substances on motor activity in porcine uterus has been studied, although the role of β3–adrenoceptors in this regulation in early pregnancy has not yet been described. Therefore, the aim of the study was to verify whether β3–adrenoceptors are involved in the regulation of the contractile activity of the porcine uterine smooth muscle in the peri-implantation period and comparison of the relaxative effect of selective β2– and β3–agonists.


MATERIAL AND METHODS

Animals

Prepubertal crossbred gilts (n = 8) with an average BW of 104 ± 4.6 kg and approximately 30 w of age were used. The gilts were treated hormonally by an i.m. injection of 750 IU of eCG (Folligon, Intervet, Holland) and 500 IU of hCG (Chorulon, Intervet) given 72 h later. Then, 13 to 16 d after hCG administration, the gilts were treated with 10 mg of dinoprost, an analog of prostaglandin F (Dinolitic; Pfizer Trading, warsaw, Poland) and 24 h later again with eCG 750 IU followed with hCG 500 IU after 72 h. Subsequently, 24 h after the last hCG administration, the gilts were inseminated twice at 12 h intervals. The d of the second insemination was the first d of pregnancy. At d 12 to 14 of pregnancy the gilts were slaughtered. To confirm pregnancy, the uterine horns were flushed with 10 mL of PBS (pH = 7.4) to determine the presence of embryos in uterine flushings (Wasielak et al., 2008). The exact number of embryos was not possible to count because of their defragmentation caused by flushing. All procedures involving animals were conducted in accordance with the rules approved by the Local Ethics Commission of the University of Warmia and Mazury in Olsztyn.

Preparation of the Uterine Strips for Measurement of Their Contraction

Segments of the uterine horns (about 1.5 cm in length), collected from the middle part of the horns were transferred to ice, moved to the laboratory, and immediately processed for examination of contractility. The contractile activity was examined according to the method described previously (Markiewicz et al., 2012; Jana et al., 2013). Briefly, the myometrial strips (3 × 5 mm) were resected, washed in saline, and mounted between 2 stainless steel hooks in a 5 mL organ bath (Schuler Organ bath type 809; Hugo Sachs Electronic, March Hugstetten, Germany) under conditions of resting tension of 10 mN. The strips were kept in an incubation solution of the following composition (mmol/L): NaCl, 120.3; KCl, 5.9; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 15.5 and glucose, 11.5; pH 7.4. The solution was maintained at 37°C and continuously saturated with a mixture of 95% O2 and 5% CO2. Measurements of the contractile activity of myometrial strips were conducted using a force transducer (HSE F-30 type 372), a bridge coupler type 570, and the graphic recording was made on a recorder (Hugo Sachs Elektronik) with the HSE-ACAD/W software.

Schedule of Contractile Activity Examination

The recording was started after prior equilibration for at least 60 to 90 min. At the beginning of the examination, the strips were incubated with increasing (10–6 to 10–4 M) concentrations of acetylcholine (ACh; Sigma, St. Louis, MO) to determine the viability of tissues and their usefulness for further study. Next, the effect of increasing concentrations (10–9 to 10–5 M) of salbutamol, a selective β2–adrenoceptor agonist (Sigma) or BRL 37344, a selective β3–adrenoceptor agonist (Tocris Bioscience, Avonmouth, Bristol, UK) given alone and in the presence of antagonists were examined. The antagonists: butoxamine (a selective β2–adrenoceptor antagonist; Sigma), propranolol (a nonselective β1– and β2–adrenoceptor antagonist, Sigma), and bupranolol (a nonselective β1–, β2–, and β3–adrenoceptor antagonist; LGC Standarts GmbH, Wesel, Germany), each at concentration of 10–4 M, were added to the organ bath 15 min before the agonists administration. Finally, at the end of treatment with examined substances to determine the viability of tissues, ACh was repeatedly administered in the same doses as given before. Only those results for which the differences in response to the stimulation by ACh at the beginning and the end of the treatment were less than 20% were included into the statistical analysis. Between each set of examinations, the tissue chambers were washed 3 times with 15 mL of the incubation solution at 10 min intervals. A graphic schedule of the treatment is shown in Fig. 1. The spontaneous contractile activity measured 10 min before the administration of the agonists or antagonists plus agonists was regarded as the control level. The responses for examined substances were quantified by calculating (i) the resting tension plus amplitude (tension; mN) which allowed for evaluation the absolute effect, (ii) the amplitude (mN), and (iii) frequency/rate of contractions (number per 10 min).

Figure 1.
Figure 1.

Diagram showing the schedule of the uterine strips treatment with acetylcholine; salbutamol, a selective agonist of β2-adrenoceptors; BRL 37344, a selective agonist of β3-adrenoceptors; butoxamine, a selective antagonist of β2-adrenoceptors; propranolol, a non-selective antagonist of β1- and β2–adrenoceptors and bupranolol, a nonselective antagonist of β1–, β2–, and β3–adrenoceptors. Concentrations of the examined substances are expressed in moles.

 

Statistical Analysis

Numerical values of the contractile activity (tension, frequency, and amplitude) of the myometrial strips before the application of the examined substances (pretreatment period) were calculated for 10 min and accepted as 100%. The results calculated for 10 min period after treatments were expressed as a percentage (mean ± SD; n = 8) of the tension, frequency, and amplitude measured in pretreatment period. The statistical significance of the differences obtained were assessed by one-way analysis of variance ANOVA (Graphpad Prism 3.1; Graphpad Software, Inc., San Diego, CA) followed by Bonferroni’s multiple comparison test. The concentration of drug required to elicit half of the maximal effect (EC50), which is expressed as the –log EC50 and known as the pD2 was calculated as it was described by Dennedy et al. (2001, 2002). The Student t test was used to compare pD2 (–log EC50) and maximal inhibitory response values. The statistical significance was considered when the probability value was P < 0.05.


RESULTS

All experiments were performed on the myometrial strips showing regular spontaneous contractile activity after prior equilibration (Fig. 2A) and an increase in tension after ACh administration.

Figure 2.
Figure 2.

Representative diagrams showing contractile activity of the porcine myometrial strips collected on d 12–14 of pregnancy; (A) spontaneous, regular activity of untreated strips; (B) the effect of increasing (10–9 to 10–5 M) concentrations of salbutamol (SAL); and (C) the effect of increasing (10–9 to 10–5 M) concentrations of BRL 37344 (BRL).

 

Effect of Salbutamol and BRL 37344

The changes in the contractile activity in response to salbutamol and BRL 37344 treatment are shown in Fig. 2BC. The decrease in the tension (Fig. 3A), frequency (Fig. 3B) and amplitude (Fig. 3C) after salbutamol administration at concentrations 10–8 to 10–5 M, 10–7 to 10–5 M, and 10–5 M was significantly greater, respectively, when compared to BRL 37344 treatment.

Figure 3.
Figure 3.

The effect of increasing (10–9 to 10–5 M) concentrations of salbutamol (Sal), a selective agonist of β2-adrenoceptors and BRL 37344 (BRL), a selective agonist of β3-adrenoceptors on the tension (A), frequency (B), and amplitude (C) of contractions of the porcine myometrial strips collected in the peri-implantation period (d 12 to 14 of the pregnancy). The results calculated for a 10 min period after treatments were expressed as a percentage (mean ± SD; n = 8) of the tension, amplitude, and frequency determined for a 10 min period before agonists administration and accepted as 100% (solid horizontal line). #P < 0.05; ##P < 0.01; ###P < 0.001 indicate significant differences compared to 10 min pretreatment period. **P < 0.01; ***P < 0.001 indicate significant differences between agonists used at the same concentrations.

 

The calculated pD2 value and the mean maximal inhibition achieved for salbutamol and BRL 37344 are shown in Table 1. Salbutamol exerted a significantly (P < 0.001) more potent reduction of the tension (pD2 = 6.98 ± 0.93 (SD)) than BRL 37344 (pD2 = 3.41 ± 0.1.61).


View Full Table | Close Full ViewTable 1.

pD2 values and maximum contractile inhibitory effects of salbutamol and BRL 373441

 
pD2 Maximum inhibition (%)
Salbutamol Tension 6.98 ± 0.93 33.86 ± 7.38
Frequency 6.74 ±0.32 5.51 ± 4.51
Amplitude 6.66 ± 0.37 16.01 ± 7.78
BRL 37344 Tension 3.41 ± 1.61*** 57.85 ± 19.56
Frequency 5.78 ± 0.47 28.68 ± 11.26
Amplitude 5.67 ± 0.66 36.11 ± 5.72
1Data are expressed as mean ± SD (n = 8).
***Indicate significant (P < 0.001) difference for pD2 calculated for the tension after salbutamol and BRL 37344 treatment.

There were no significant differences between calculated pD2 values for frequency and amplitude, or mean maximal inhibition between both agonists.

Effect of Butoxamine Pretreatment on Salbutamol and BRL 37344 Activity

Salbutamol, administered further to butaxamine, did not exert a significant impact on tension (Fig. 4A), while triggering a significant reduction of the frequency at concentrations of 10–7 to 10–5 M (Fig. 4B) and of the amplitude at a concentration of 10–5 M (Fig. 4C) as compared to the pretreatment period. BRL 37344, administered further to butaxamine, caused a reduction of the tension (Fig. 4A) and frequency (Fig. 4B) at concentrations of 10–6 to 10–5 M and of the amplitude at the concentration of 10–5 M (Fig. 4C).

Figure 4.
Figure 4.

The influence of butoxamine (But; 10–4 M), a selective antagonist of β2-adrenoceptors on changes in the tension (A), frequency (B), and amplitude (C) induced by increasing (10–9 to 10–5 M) concentrations of salbutamol, a selective agonist of β2-adrenergic receptors or BRL 37344 (BRL), a selective agonist of β3-adrenoceptors in the porcine myometrial strips collected in the peri-implantation period. The results calculated for a 10 min period after treatments were expressed as a percentage (mean ± SD; n = 8) of the tension, amplitude, and frequency determined for a 10 min period before butoxamine and salbutamol or BRL 37344 administration and accepted as 100% (solid horizontal line). #P < 0.05; ##P < 0.01; ###P < 0.001 indicate significant differences compared to 10 min pretreatment period. *P < 0.05; **P < 0.01; ***P < 0.001 indicate significant differences between agonists used at the same concentrations.

 

BRL 37344 decreased the tension (Fig. 4A) and amplitude (Fig. 4C) significantly more at concentrations 10–6 to 10–5 M and 10–5 M, respectively, and decreased the frequency significantly lower at concentration 10–9 M when compared to salbutamol.

Effect of Propranolol on Salbutamol and BRL 37344 Activity

Salbutamol, administered further to propranolol, did not have any significant impact on tension (Fig. 5A) or frequency (Fig. 5B), although it substantially reduced the amplitude at the concentration of 10–5 M (Fig. 5C) as compared to the pretreatment period. In contrast, BRL 37344, when administered further to propranolol, triggered a reduction in tension (Fig. 5A) and frequency (Fig. 5B) at the concentration of 10–5 M and in the amplitude at concentrations of 10–7 to 10–5 M (Fig. 5C).

Figure 5.
Figure 5.

The influence of propranolol (Pro; 10–4 M), an antagonist of β1– and β2–adrenoceptors on changes in the tension (A), frequency (B), and amplitude (C) induced by increasing (10–9 to 10–5 M) concentrations of salbutamol, a selective agonist of β2-adrenergic receptors or BRL 37344 (BRL), a selective agonist of β3-adrenoceptors in the porcine myometrial strips collected in the peri-implantation period. The results were calculated for a 10 min period after treatments were expressed as a percentage (mean ± SD; n = 8) of the tension, amplitude and frequency determined for a 10 min period before propranolol and salbutamol or BRL 37344 administration and accepted as 100% (solid horizontal line). #P < 0.05; ##P < 0.01; ###P < 0.001 indicate significant differences compared to 10 min pretreatment period. *P < 0.05; **P < 0.01 indicate significant differences between agonists used at the same concentrations.

 

BRL 37344 decreased the tension (Fig. 5A), frequency (Fig. 5B), and amplitude (Fig. 5C) significantly more at concentrations 10–5 M, 10–7 M, and 10–5 M, respectively, when compared to salbutamol.

Effect of Bupranolol on Salbutamol and BRL 37344 Activity

Salbutamol, administered further to bupranolol, did not cause any significant changes in tension (Fig. 6A) while it reduced the frequency at concentrations of 10–6 to 10–5 M (Fig. 6B) and amplitude at a concentration of 10–5 M (Fig. 6C) as compared to the pretreatment period. BRL 37344, administered further to bupranolol, did not have any significant effect on any of the investigated parameters (Fig. 6) as compared to the pretreatment period. BRL 37344 decreased the tension (Fig. 6A) significantly more at concentrations 10–9 to 10–6 M but decreased the frequency (Fig. 5B) and amplitude (Fig. 5C) significantly lower at concentrations 10–7 to 10–5 M and 10–5 M, respectively, as compared to salbutamol.

Figure 6.
Figure 6.

The influence of bupranolol (Bup; 10–4 M), an antagonist of β1–, β2–, and β3– adrenoceptors on changes in the tension (A), frequency (B), and amplitude (C) induced by increasing (10–9 to 10–5 M) concentrations of salbutamol, a selective agonist of β2-adrenergic receptors or BRL 37344 (BRL), a selective agonist of β3-adrenoceptors in the porcine myometrial strips collected in peri-implantation period. The results calculated for a 10 min period after treatments were expressed as a percentage (mean ± SD; n = 8) of the tension, amplitude and frequency determined for 10 min period before bupranolol and salbutamol or BRL 37344 administration and accepted as 100% (solid horizontal line). #P < 0.05; ##P < 0.01; ###P < 0.001 indicate significant differences compared to 10 min pretreatment period. *P < 0.05; **P < 0.01; ***P < 0.001 indicate significant differences between agonists used at the same concentrations.

 


DISCUSSION

Since it is generally accepted that β2–adrenoceptors are inhibitory in nature, their agonists such as ritodrine and salbutamol are used clinically in the treatment of preterm labor (de Heus et al., 2009; Motazedian et al., 2010; Parida et al., 2013). Recently, an increased interest in the role of β3–adrenoceptors in the regulation of myometrium contractile activity in human (Bardou et al., 2000, 2007; Dennedy et al., 2001, 2002; Rouget et al., 2005; Pędzińska-Betiuk et al., 2011), rat (Yurtcu et al., 2006; Clouse et al., 2007; Minorics et al., 2009), and mouse (Parida et al. (2013) has been observed. The results of the above studies have demonstrated the species variability of β3–adrenoceptor pharmacology, as well as the heterogeneous responsiveness to β3–adrenoceptor stimulation. Moreover, in gilts there are differences in myometrial activity between cyclic and pregnant gilts (Kitazawa et al., 2001; Markiewicz et al., 2016). According to our knowledge, the role of β3–adrenoceptor in the contractile activity of the porcine myometrium in early pregnancy has not been examined.

The contractile activity of the myometrium has been described by many parameters, e.g., mean amplitude of contraction (Dennedy et al., 2002), areas under the curves (Minorics et al., 2009), amplitude, frequency, and basal tension (Pędzińska-Betiuk et al., 2011). In our study, we have focused on changes in resting tension plus amplitude (called tension) which allowed evaluation of the absolute effect of substances examined as well as amplitude and frequency/rate of contractions. Using these 3 parameters we were able to describe more precisely changes in the contractile activity. The findings from our study strongly demonstrate that both salbutamol and BRL 37344 decreased the tension, amplitude, and frequency of contraction in myometrial strips collected from gilts in the peri-implantation period, but these changes were more evident after salbutamol treatment. This study also reports that salbutamol exerts its effect on tension by acting on the β2–adrenoceptor without any obvious effect on β1– and β3–adrenoceptors. In contrast, BRL 37344 action was mediated through the β3–adrenoceptor, with possible additional effects on β1– and β2–adrenoceptors. This suggestion results from the fact that blockage of β2–adrenoceptors with butoxamine significantly inhibited the relaxing effect of salbutamol, mainly by preventing a decrease in the tension and less evident decrease in frequency and amplitude. This antagonist, however, changed the effect of BRL 37344 in a different way, as the reduction of tension was more pronounced, while a decrease in amplitude was lower in contrast to the administration of the agonist alone. In myometrial strips pre-incubated with propranolol, a slight reduction of amplitude was only noted following the administration of salbutamol. When BRL 37344 was administered further to propranolol, this agent caused a substantial reduction of the investigated parameters, although these changes were less pronounced compared to the effect of this agonist used alone. These findings confirm that the relaxing effect of salbutamol is a result of its impact mainly on the β2–adrenoceptors, whereas the activity of BRL may also involve the other β-adrenoceptors. These data are consistent with previous studies demonstrating that the β2–adrenoceptors are a dominant subtype, and their stimulation results in inhibiting the contractility of myometrium in sexually mature gilts (Kitazawa et al., 2001). Furthermore, it has been reported that propranolol and BRL 37344 demonstrate affinity to α-adrenergic receptors in the rat aorta (Brahmadevara et al., 2004; Leblais et al., 2004). Kitazawa et al. (2000) showed that α2–adrenoceptors stimulation caused contraction of the longitudinal muscles in porcine myometrium. Therefore, the binding of these substances with α-adrenoceptors can change the response to BRL 37344 stimulation. The existence of an additional mechanism of BRL 37344 action, which is not related to the activation of the β3–adrenoceptors, was also postulated by Pędzińska-Betiuk et al. (2011), who described human nonpregnant myometrium contractility after co-administration of propranolol with BRL 37344. In our study, bupranolol completely abolished the relaxative effect of BRL 37344, which confirmed the existence of β3–adrenoceptors in the porcine myometrium in the peri-implantation period. Bupranolol also eliminated the relaxing effect of salbutamol, although this was mainly due to the blockage of β2–adrenoceptors. A similar antagonistic impact of bupranolol on the effect of BRL 37344 and ritodrine (β2–adrenoceptors agonist) was observed in human nonpregnant myometrium (Pędzińska-Betiuk et al., 2011).

In conclusion, the results indicate that β3–adrenoceptors are involved in the relaxation of the porcine myometrium in the peri-implantation period. Moreover, the data show that both examined agonists effectively relax the porcine myometrium, although BRL 37344 is less potent than salbutamol. Pharmacologic modulation of uterine β3–adrenoceptors may be used to reduce excessive systolic function during embryo transfer and pregnancy risk. However, the exact explanation of the physiological role of β3–adrenoceptors in the porcine uterus requires further studies.

 

References

Footnotes


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