[The effect of the ARC 239 on the myometrial and cervical action in the rat, in vitro]. / Az ARC 239 hatása a miometrium és a cervix muködésére patkányban, in vitro.

The premature labour is one of the major challenges in the clinical practice. Finding new agents and mechanisms in the control of uterine activity is the main objective of the last decade's experiments. One of the new targets is the alpha2-adrenoceptors (alpha2-AR). The purpose of this study was to determine the effect of the alpha2B/C-adrenoceptor blocker ARC 239 on the myometrial contractions and the cervical resistance on pregnant rats, in vitro. We identified the alpha2-adrenoceptor subtypes proteins both in the myometrial and the cervical samples. In isolated organ studies, the ARC 239 exerted a strong inhibitory effect on noradrenaline-stimulated contractions. The effect of ARC 239 on labour-induced myometrial samples was also convincing. In the stretching test, the cervical resistance was increased and decreased in by ARC 239 on pregnancy days 18 and 20, respectively. ARC 239 did not have effect on the 22-day pregnant cervical samples. These results were supported by the cAMP studies. We can conclude that, the alpha2B-adrenoceptors predominate and mediate contraction, while the alpha2A- and alpha2C-ARs decrease the contractile response to noradrenaline in 22-days-pregnant animals. In the pregnant cervix the alpha2-adrenoceptors can couple to both G(i)- and G()-proteins in the 18- and 20-day-pregnant samples, respectively, resulting in increase or decrease in the cervical resistance. Based on these facts we suggest that ARC 239 may open new perspective in the influence of premature labour.

Gestagen treatment enhances the tocolytic effect of salmeterol in hormone-induced preterm labor in the rat in vivo.

OBJECTIVE: The purpose of this study was to determine whether gestagen treatment enhances the effects of beta2-mimetics in hormone-induced preterm delivery in pregnant rats in vivo. STUDY DESIGN: Preterm birth was induced with a combination of mifepristone and prostaglandin E2 on day 19 of pregnancy. The animals were treated with salmeterol or gestagens (progesterone or 17alpha-hydroxyprogesterone) or their combination. The treatments were launched on different days (15-18) of pregnancy. The efficacy of treatment was determined in terms of the delivery time counted from the mifepristone injection. RESULTS: Salmeterol treatment delayed premature labor by 2.4 hours, whereas the delay because of the gestagen-salmeterol combinations was more than 5 hours. Progesterone had no effect on the delivery time. CONCLUSION: Parallel treatment with salmeterol and gestagens can be more than twice as effective as salmeterol therapy alone. These results open up a possibility for human trials of combined beta2-agonist-gestagen therapy in threatening preterm delivery.

Different roles of alpha2-adrenoceptor subtypes in non-pregnant and late-pregnant uterine contractility in vitro in the rat.

The roles of the alpha2-adrenoceptor (alpha2-AR) subtypes (alpha2A-, alpha2B- and alpha2C-AR) in uterine contractility have not been investigated. The aims of this study were to identify these receptors in the non-pregnant and the late-pregnant rat myometrium and to determine their roles in contractions. We found that the myometrial alpha2-AR subtypes are involved differently in the control of late-pregnant contractions, while they have no influence on the contractions of the non-pregnant myometrium. The myometrial expressions of the alpha2-AR subtypes were determined by RT-PCR and Western blotting techniques. In vitro contractions were stimulated with noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (alpha2A), ARC 239 (alpha2B/C) and spiroxatrine (alpha2C). cAMP production was followed by noradrenaline stimulation in the presence of isobutylmethylxanthine and forskolin, and alterations induced in it by the antagonists were determined with an Enzyme Immunoassay Kit. The most effective antagonist was tested on labour-induced uteri in vitro. All the alpha2-AR subtypes were identified in both non-pregnant and pregnant uteri. Noradrenaline was not able to contract the non-pregnant tissue in the presence of propranolol and doxazosin, while its contracting effect in the pregnant uteri was enhanced by BRL 44408, spiroxatrine and the combination BRL 44408+spiroxatrine. ARC 239 exerted a strong inhibitory effect on noradrenaline-stimulated contractions. The increasing and the decreasing effects of the compounds were confirmed by the changes in the intracellular cAMP levels. The effect of ARC 239 on the labour-induced myometrium was similar to that on the 22-day-pregnant myometrium. The stimulation of alpha2-ARs does not evoke contractions in the non-pregnant uterus. The alpha2A- and alpha2C-ARs mediate decreases, while the alpha2B-AR mediates an increase in the contractions in the 22-day-pregnant myometrium. These differences may offer new targets for drugs against premature contractions in pregnancy.

Effect of alpha-adrenoceptor subtype-selective inverse agonists on non-pregnant and late-pregnant cervical resistance in vitro in the rat.

1. The aim of the present study was to compare and elucidate the effects of alpha(1)-adrenoceptor (alpha(1)-AR) subtype-selective inverse agonists on non-pregnant and late-pregnant rat cervical tone. 2. Cervical resistance was investigated in in vitro stretching tests in the absence or presence of alpha(1)-AR subtype-selective inverse agonists (WB 4101, AH 11110A and BMY 7378; all at 10(-6) mol/L), whereas the mRNA levels and density of the alpha(1)-AR subtypes and the G-protein-activating effects of the inverse agonists were determined by reverse transcription-polymerase chain reaction, western blot and [(35)S]-GTPgammaS binding techniques, respectively. 3. The inverse agonists did not cause any change in resistance in non-pregnant and 18-day-pregnant samples. WB 4101 increased cervical resistance from Day 20, whereas AH 11110A had no effect and BMY 7378 exhibited such an action only on Day 21. Phenylephrine (10(-4) mol/L) had no effect on cervical resistance on Day 22. The mRNA levels and density of all alpha(1)-AR subtypes were increased on Day 18, but no further changes were observed after that. The [(35)S]-GTPgammaS binding studies revealed increased G-protein activation of alpha(1A)-AR and a moderate G-protein activation of alpha(1B)- and alpha(1D)-AR. The effect of WB 4101 to increase [(35)S]-GTPgammaS binding was blocked by pertussis toxin (50 ng/mL). Phenylephrine caused a slight and significant decrease in the amount of activated G-protein on Day 22. 4. The effects of inverse agonists on the alpha(1A)-AR can enhance cervical resistance in the late-pregnant rat in vitro. This action is mediated, at least in part, by a pertussis toxin-sensitive G(i)-protein. This effect of the alpha(1A)-AR inverse agonist WB 4101 may offer a new therapeutic target in the prevention of premature labour.

Pregnancy-induced decrease in the relaxant effect of terbutaline in the late-pregnant rat myometrium: role of G-protein activation and progesterone.

The effectiveness of beta2-agonists in preterm delivery is reduced by several factors. The aim of this study was to determine the influence of late pregnancy in the uterus-relaxing effect of terbutaline in the rat in vitro. Rat uterine tissues from late pregnancy (days 15, 18, 20 and 22) were used. In vitro electrical field-stimulation (EFS) was used to evoke contractions. The radioligand-binding technique, reverse transcription-polymerase chain reaction and radioimmunoassay technique were used to determine the beta-adrenergic receptor density and mRNA level and the plasma sex hormone level, respectively. The activated G-protein level of the beta-adrenergic receptors was investigated by a radiolabelled GTP binding assay.EFS-induced contractions were inhibited by terbutaline. This effect decreased towards term with respect to both the EC50 and maximal inhibition values. A drop in plasma progesterone level was also detected. Binding studies revealed an increase in beta-adrenergic receptor number on the last day of pregnancy, which correlated with the change in receptor mRNA level. The G-protein-activating effect of terbutaline decreased continuously between days 15 and 20. Surprisingly, terbutaline decreased the G-protein activation to below the basal level on day 22. However, progesterone pretreatment set back the uterine action of terbutaline, increased the density of the beta2-adrenergic receptors and their mRNA level and increased the G-protein-activating property of terbutaline. These data provide evidence of a pregnancy-induced decrease in activated G-protein level after beta2-agonist stimulation. The decrease in plasma progesterone level has a crucial role in this process. The effects of beta2-adrenergic receptor agonists in tocolytic therapy may possibly be potentiated with progesterone.

Terbutaline increases the cervical resistance of the pregnant rat in vitro.

Cervical ripening is a crucial process leading to delivery. Early dilation of the pregnant cervix can contribute to premature labour. The maturity of the cervix can be characterized by its resistance to mechanical stretching. Although a number of compounds are considered to increase cervical resistance (e.g., progesterone, nitric oxide synthase inhibitors and nonsteroidal anti-inflammatory drugs), none of them seem to be safe for clinical application. Other compounds, such as beta(2)-adrenergic receptor (beta(2)-AR) agonists, have been used for several decades to stop premature myometrium contractions, but their cervical action has never been investigated. The aim of this study was to detect the effects of the beta(2)-AR agonist terbutaline on nonpregnant and late-pregnant (day 18, 20, 21 or 22) cervices isolated from Sprague-Dawley rats. Cervical resistance was measured by means of a mechanical stretching test in vitro, the beta(2)-AR density was determined by Western blot analysis, the beta(2)-AR mRNA was determined by RT-PCR, while the G-protein activation following cervical beta(2)-AR stimulation with terbutaline was evaluated via a [(35)S]GTPgammaS binding assay. Terbutaline at 10(-6) M increased the cervical resistance of the late-pregnant samples in vitro from day 18 to day 22, but did not alter the resistance of the nonpregnant samples. This cervical resistance-increasing effect was concentration dependent and antagonized with propranolol on day 21. Terbutaline was ineffective on cervical samples when gradual stretching was omitted. RT-PCR and Western blot studies revealed increased beta(2)-AR mRNA and beta(2)-AR levels respectively on day 18 of pregnancy compared with the nonpregnant cervix, but no further changes were detected up to the end of pregnancy. The [(35)S]GTPgammaS binding assay demonstrated a decreased G-protein activation on the days of pregnancy investigated, but no activation was found in the nonpregnant samples. The degree of decrease in G-protein activation by terbutaline was in harmony with its cervical resistance-increasing action. On day 21, the G-protein activation-decreasing effect of terbutaline was antagonized with propranolol. We presume that the cervical resistance-increasing effect of terbutaline is a consequence of its G-protein activation-decreasing property via beta(2)-ARs, which finally leads to an increased muscle resistance against mechanical stretching. This action of terbutaline seems unique among the smooth muscles, and may open up a new perspective in the prevention of premature labour. Clinical experience indicates that beta(2)-AR agonists will not be sufficient to stop the overall process, but their combination with more potent inhibitors of uterine contractions may be of clinical benefit.