PRACE ORYGINALNE Ginekol Pol. 2011, 82, 918-924 ginekologia Differences in the effects ofβ 2- and β3- adrenoceptor agonists on spontaneous contractions of human nonpregnant myometrium Odmienny wpływ agonistów receptorów β2- i β3-adrenergicznych na spontaniczne skurcze myometrium kobiet nieciężarnych Pędzińska-Betiuk Anna1*, Modzelewska Beata1, Jóźwik Marcin2, Jóźwik Maciej3, Kostrzewska Anna1 1 Department of Biophysics, Medical University of Białystok, Białystok, Poland 2 Department of Gynecology and Obstetrics, Kliniken Nordoberpfalz, Akademisches Lehrkrankenhaus der Universität Regensburg, Weiden, Germany 3 Department of Gynecology, Medical University of Białystok, Białystok, Poland * Current address: Department of Experimental Physiology and Pathophysiology, Medical University of Białystok, Białystok, Poland Abstract Objective: This study aimed to compare the relaxant properties of BRL 37344 with β2-adrenoceptors agonist ritodrine on the contractility of human nonpregnant myometrium. Material and methods: The activity of myometrial strips mounted in an organ bath was recorded under isometric conditions using force transducers with digital output. Contractility before and after cumulative additions of both uterorelaxants and with preincubation with β-adrenoceptor antagonists bupranolol, propranolol, and butoxamine were studied. Results: Both BRL 37344 (10-10 – 10-4 mol/L) and ritodrine (10-10 – 10-5 mol/L) decreased the area under curve, or AUC, value (logIC50 -6.45 ± 0.18 and -8.71 ± 0.35, respectively), and the degree of inhibition of spontaneous contractile activity was similar (< 30%). However, BRL 37344 decreased the mean frequency of contractions, whereas ritodrine decreased the mean amplitude of contractions. The inhibition of contractions by BRL 37344 was partially antagonized by bupranolol and propranolol, but not with butoxamine. The inhibition by ritodrine was counteracted by all these antagonists. Conclusions: The effects of BRL37344 and ritodrine on human nonpregnant myometrium are quantitatively similar in respect to the inhibition of spontaneous contractility, yet are also distinct due to their substantially different influences on contraction parameters. Our data indicate thatβ 3-adrenoceptor activation is not the sole effect of BRL 37344 on this tissue. Key words: adrenergic agonists / BRL 37344 / human / myometrium / ritodrine / Adres do korespondencji Anna Kostrzewska Department of Biophysics, Medical University of Bialystok, Mickiewicza 2A, 15-230 Białystok, Poland Otrzymano: 30.09.2011 tel.: +48 85 748 56 67 Zaakceptowano do druku: 14.11.2011 e-mail: [email protected] 918 Nr 12/2011 Ginekol Pol. 2011, 82, 918-924 PRACE ORYGINALNE ginekologia Pędzińska-Betiuk A, et al. Streszczenie Cel pracy: Niniejsze badanie zaprojektowano celem oceny rozkurczowego działania BRL 37344 w porównaniu do ritodryny (referencyjnego agonisty receptorów β2-adrenergicznych) na spontaniczną czynność skurczową ludzkiego nieciężarnego myometrium. Materiał i metody: Skurcz izolowanych skrawków mięśni gładkich rejestrowano w warunkach izometrycznych przy użyciu cyfrowych przetworników siły. Ocenie poddawano zmiany kurczliwości skrawków podczas kumulacyj- nego zwiększania stężeń agonistów bez obecności oraz w obecności antagonistów receptorów β adrenergicznych: bupranololu, propranololu oraz butoksaminy. Wyniki: Inkubacja skrawków myometrium z BRL 37344 (10-10 – 10-4 mol/L) oraz ritodryną (10-10 – 10-5 mol/L) skutkowała porównywalnym (<30%) osłabieniem ich kurczliwości przejawiającym się spadkiem wartości pola po- wierzchni pod krzywą, czyli AUC (odpowiednio, logIC50 -6,45 ± 0,18 oraz -8,71 ± 0,35). W odróżnieniu od ritodryny, której efekt działania polegał na obniżaniu średniej amplitudy skurczu, inkubacja z BRL 37344 zmniejszała średnią częstotliwość skurczu. Działanie rozkurczowe BRL 37344 było częściowo hamowane przez propranolol oraz bu- pranolol, podczas gdy wpływ ritodryny był znoszony przez wszystkich zastosowanych antagonistów. Wnioski: Porównywalne ilościowo działanie rozkurczowe BRL 37344 oraz ritodryny na spontaniczną czynność skurczową ludzkiego myometrium jest wynikiem odmiennego wpływu tych agonistów na poszczególne parametry skurczowe. Nasze dane wskazują, iż aktywacja β3-adrenoceptorów nie jest jedynym kierunkiem działania BRL 37344 na tę tkankę. Słowa kluczowe: agoniści receptorów adrenergicznych / BRL 37344 / człowiek / / myometrium / ritodryna / Introduction Material and methods Both receptors subclasses, β2- and β3-adrenoceptors have Human myometrial tissue been shown to mediate the contractile response of the human Human uterine tissues were collected from 20 nonpregnant, uterus. The regulation of the β-adrenoceptors in humans may premenopausal women (aged 42-50 years, median 48 years) vary due to numerous pharmacologic, physiologic, and hormonal undergoing hysterectomy for benign gynecological disorders, influences, and the impact of the phases of the menstrual cycle, such as dysfunctional bleedings and cervical pre-malignancy, pregnancy, or menopause [1]. Furthermore, the β3-adrenoceptors and being operated upon in the follicular phase of their menstrual have been characterized and their functionality shown in pregnant cycles. All patients gave informed consent to the study which had human myometrium [2, 3]. The expression of β3-adrenoceptors been approved by the Ethics Committee of the Medical University increases in the near-term myometrium [1]. Two populations of of Białystok (approval number R-I – 003/307/2005). binding sites corresponding to β2- and β3-adrenoceptors have Myometrial samples were excised transversely from been identified, and the predominance of the β3-adrenoceptors the uterine fundus, placed in ice-cold Tyrode’s solution, and subtype in the nonpregnant and pregnant human myometrium immediately transferred to the laboratory where they were were also confirmed [4]. processed as previously described [6]. Briefly, 4-8 strips, 6-7 β2-Adrenoceptor agonists, such as ritodrine, are well-known mm in length and 2x2 mm in cross-sectional area were obtained drugs used for the management of preterm labor. However, under a dissecting microscope. The strips were then mounted in maternal and fetal cardiovascular side effects restrict their an organ bath containing 20 mL of Tyrode’s solution at 37 ºC, pH limitations in clinical practice [5]. According to our previous 7.4, and bubbled with carbogen (95% O2 + 5% CO2). Tyrode’s study, a β3-adrenoceptors agonist, CL 316243, efficiently inhibits solution consisted of (mmol/L): NaCL 139.6; KCl 2.68; MgCl2 spontaneous contractions of human nonpregnant myometrium 1.05; NaH2PO4 1.33; CaCl2 1.80; NaHCO3 25.0; and glucose [6]. This finding is parallel to the Rouget et al. study, in which 5.55. The strips were left in the organ bath for equilibration a β3-adrenoceptors agonist SR 59119A was shown to diminish period of 1-2 hours, during which time the passive tension was spontaneous rhythmic contractile activity in this tissue [4]. adjusted to 2 mN. After equilibration, regular phasic contractions The influence on the contractile activity of another interesting were achieved. β3-adrenoceptors agonist, BRL 37344, has been studied in the The activity of the myometrium was recorded under isometric pregnant human myometrium [3, 7]. Some reports, indicating a conditions by means of force transducers with digital output. BRL marked relaxing effect of BRL 37344 on the nonpregnant uterine 37344 or ritodrine was added cumulatively to the organ chambers smooth muscle, were based on experiments on rats and pigs [8, (bath concentrations in the range 10-10 mol/L – 10-4 mol/L and 10- 9]. Surprisingly, there is virtually no information available on the 10 mol/L – 10-5 mol/L, respectively) at 15-minute intervals, and agent’s effects on human nonpregnant uterus. the effects were recorded. The level of spontaneous contractile Therefore, taking into account the species variability of β3- activity before the addition of agonist was regarded as the control adrenoceptor pharmacology investigated [10], the purpose of the level. The responses were quantified by: calculating the area present study was to compare the ability of BRL 37344 to relax under the curve (AUC) of active contraction [11], the amplitude, spontaneous contractility of the human nonpregnant myometrium frequency of the contractions, and the basal tension. The AUC with that of ritodrine. was measured from basal tension over a 10-minute period before a further addition of β-adrenoceptor agonist. © Polskie Towarzystwo Ginekologiczne 919 PRACE ORYGINALNE Ginekol Pol. 2011, 82, 918-924 ginekologia Differences in the effects ofβ 2- and β3-adrenoceptor agonists on spontaneous contractions of human nonpregnant myometrium. Effect of preincubation with β-adrenoceptor antagonists statistically with PRISM 3 using Student’s t-test and a two- or The tissue responses were evaluated by comparing them with one-way ANOVA, where appropriate. The statistical significance those obtained with the antagonist, before the administration of was considered when the probability value (P) was <0.05. the first concentration of BRL 37344 or ritodrine. Butoxamine (β - 2 Results adrenoceptor antagonist), propranolol (β1- and β2-adrenoceptor antagonist), and bupranolol (nonselective adrenoceptor All experiments were performed on myometrial strips antagonist), each at concentration of 10-6 mol/L, were added to exhibiting regular spontaneous contractile activity after the organ bath 20 minutes before the agonists administration. equilibration. The mean