[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.

The roles of alpha2-adrenoceptor subtypes in the control of cervical resistance in the late-pregnant rat.

The roles of the alpha(2)-adrenoceptor subtypes in the regulation of cervical resistance have previously not been investigated. The aim of the present study was to identify these receptors in the late-pregnant cervix and determine their functions in vitro in the rat. The expressions of the alpha(2)-adrenoceptor subtypes were determined by means of RT-PCR and Western blotting techniques. The changes in cervical resistance due to subtype-selective antagonists were investigated in stretching tests. The cyclic AMP immunoassay technique was used to detect the level of cyclic AMP following stimulation of the alpha(2)-adrenoceptors with or without pertussis toxin. On pregnancy days 18, 20, 21 and 22, the RT-PCR and Western blotting studies revealed the expressions of all three alpha(2)-adrenoceptor subtype mRNAs and proteins. On days 18 and 20, noradrenaline increased and decreased the resistance, respectively. Its effect was blocked by each of the antagonists used, except ARC 239 on both days. On day 21, noradrenaline again increased the resistance, this effect being maintained only in the presence of spiroxatrine. Noradrenaline was ineffective on day 22. These results were supported by the changes in cyclic AMP levels. Pertussis toxin pretreatment eliminated the changes in the cyclic AMP level on days 18 and 21. We presume that the alpha(2A)- and alpha(2C)-adrenoceptors play predominant roles in the regulation of cervical resistance on days 18-21. Depending on the day of pregnancy, stimulation of these alpha(2)-adrenoceptors could even result in opposite effects. This fluctuation can be explained by the changes in the G(i)/G(s)-coupling of the alpha(2A)- and alpha(2C)-adrenoceptors.

Inflammatory processes enhance cAMP-mediated uterus relaxation in the pregnant rat: the role of TNF-alpha.

The objective of this study was to assess the in vitro uterus relaxing potency of beta(2)-adrenergic receptor (beta(2)-AR) agonists in pregnant rats after in utero administration of the bacterial lipopolysaccharide, Escherichia coli endotoxin (LPS). The LPS (100 microg/kg) was injected into the uterine lumen on day 16 of pregnancy. The effects of beta(2)-AR agonist terbutaline was tested in vitro, in isolated uterine rings precontracted by electric field stimulation. Uterine beta(2)-AR densities were detected by radioligand binding assay, the activated G-protein levels were investigated by a radiolabelled GTP binding assay. Uterine cAMP accumulation and the serum tumor necrosis factor-alpha (TNF-alpha) levels were measured by enzyme immunoassay. The endotoxin-evoked preterm delivery occurred on day 21. Higher pD(2) values of terbutaline (p < 0.001) were detected in endotoxin-treated rats: 9.14 +/- 0.36 vs. 7.71 +/- 0.12 compared with sham-operated rats. The densities or the equilibrium dissociation constants of beta(2)-ARs were not different (p > 0.05) in LPS-treated vs. control animals. Serum TNF-alpha level rose threefold after LPS treatment, but this rise was abolished by thalidomide. In LPS + thalidomide-treated rats, the effect of terbutaline became similar to that in sham-operated controls. By the measurement of myometrial cAMP levels, we documented that the concentration-response curve of terbutaline on cAMP accumulation was shifted to the left in the LPS-treated rats, with a significant rise in the pD(2). We concluded that in the case of uterine inflammation, the in vitro uterus-relaxing potency of beta(2)-agonists enhances, which is possibly mediated by TNF-alpha and uterine cAMP levels and that may serve as a rationale for the use of beta(2)-AR agonists in the attenuation of preterm uterine contractions on an inflammatory basis.

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.