Elsevier

Life Sciences

Volume 87, Issues 23–26, 18 December 2010, Pages 733-737
Life Sciences

Improving the relaxing effect of terbutaline with phosphodiesterase inhibitors: Studies on pregnant rat uteri in vitro

https://doi.org/10.1016/j.lfs.2010.10.010Get rights and content

Abstract

Aims

Previous results by our group showed that the in vitro uterus-relaxing potency of β2-adrenergic receptor (β2-AR) agonists and uterine cAMP accumulation are enhanced in case of visceral inflammation. Our aim was to study the effects of the non-selective phosphodiesterase (PDE) inhibitor theophylline and the selective PDE4 inhibitor rolipram on the uteri of intact late-pregnant female rats (on days 20 and 22 of pregnancy) and of pregnant rats treated with lipopolysaccharide (LPS) to evoke preterm labor (on day 20).

Main methods

The effects of theophylline and rolipram alone and of rolipram with terbutaline were investigated in isolated organ system. Contractions were evoked with KCl. The forskolin- and terbutaline-stimulated cAMP accumulations were determined by enzyme immunoassay, with or without rolipram.

Key findings

The maximum uterus-relaxing effects of theophylline and rolipram decreased significantly (p < 0.05) with the progression of pregnancy in intact rats. The most pronounced effect of rolipram was detected in rats challenged with LPS on day 20. Rolipram increased the in vitro effect of terbutaline both in intact and in LPS-treated rats. In the presence of rolipram, the forskolin- and terbutaline-stimulated cAMP accumulations were higher in LPS-treated than in intact rats.

Significance

The previous findings led us to conclude that the combined administration of PDE4 inhibitors with β2-agonists is of therapeutic value for the inhibition for uterine contractions, especially in the case of genital inflammation, which often triggers preterm birth. Combination therapy in general is associated with lesser side-effects, as a consequence of lower effective doses of each drug.

Introduction

Preterm uterine contractions can develop as a result of several pathological processes, among which intrauterine infection plays a key role (Romero et al., 1988). The inflammatory processes trigger a wide range of uterus-contracting factors, which eventually lead to the development of uterine contractions (Lindström and Bennett, 2005). At the same time, the contractions in the pregnant myometrium are counterbalanced in part by cyclic adenosine-monophosphate (cAMP)-dependent relaxation mechanisms. A plethora of ligands such as β2-adrenergic receptor (β2-AR) agonists can stimulate the generation of cAMP in the myometrium, whereas phosphodiesterase enzymes (PDEs) warrant termination of signalling by the degradation of cAMP to the inactive 5′-AMP (Houslay 2003).

We earlier demonstrated that the in vitro uterus-relaxing potency of the β2-AR agonist terbutaline is enhanced in pregnant rats challenged with bacterial lipopolysaccharide (LPS) to evoke preterm birth. We also found that, in case of local inflammation, terbutaline has an increased cAMP-accumulating potency, which is probably mediated by the sensitization of adenylyl cyclase by tumor necrosis factor-α (TNF-α) (Klukovits et al., 2009). On the other hand, the local PDE activity may as well be an important regulator of cAMP-dependent relaxation processes.

The PDE enzymes are encoded by 11 related gene families and are differentially expressed in human tissues (Lugnier, 2006). Human myometrial cells (Méhats et al., 1999), amniochorionic membranes (Oger et al., 2005), monocytes and neutrophils (Wang et al., 1999) express PDE4, which promotes its role in either smooth muscle or immune functions. At present, PDE4 comprises the largest PDE family, constituted by 4 genes with various alternative mRNA splices encoding at least 35 different PDE4 isoenzymes. Rolipram is the archetypal PDE4 inhibitor (Ki = 0.8 μM); and no selective inhibitor is currently available that discriminates PDE4 isoenzymes (Lugnier, 2006).

The aim of the present study was to test the effects of non-selective PDE or selective PDE4 inhibitors alone or in combination with β2-AR agonists on uterine contractions in vitro. We tested the effects of the non-selective PDE inhibitor theophylline and the specific PDE4 inhibitor rolipram on isolated uterine rings from intact and LPS-treated late-pregnant rats. We also investigated the effects of β2-AR agonist terbutaline with PDE4 blockade in the same experimental model. Uterine cAMP levels were measured by means of enzyme immunoassay (EIA) after stimulation with the direct adenylyl cyclase activator forskolin, in the presence of rolipram. The accumulation of cAMP was also detected in the presence of terbutaline and rolipram alone, and their combination.

Section snippets

Animals

The animals were treated in accordance with the European Communities Council Directives (86/609/ECC) and the Hungarian Act for the Protection of Animals in Research (XXVIII.tv.32.§). All experiments involving animal subjects were carried out with the approval of the Hungarian Ethical Committee for Animal Research (registration number: IV./01758-2/2008) and under the control of the ISO-9001:2008 Quality Management System.

Sexually mature female Sprague–Dawley rats (body mass: 140-160 g, 50–60 days

In vitro contractility studies

Uterine activity was characterized by the AUCs of KCl-stimulated contractions of uterine rings in vitro. Time control experiments revealed that KCl-stimulated contractions do not decrease significantly through the experiment (40–45 min). Although the non-specific PDE inhibitor theophylline had very limited effect in the uteri of intact rats, it showed significant effect in LPS-treated rats (Fig. 1). In the intact rats, the effect of theophylline varied, depending on the day of pregnancy: the

Discussion

Most of the interventions intended to reduce preterm birth rate have not achieved consistent benefit. Apart from manifest infections in late-pregnancy, when the termination of pregnancy is of utmost importance to save the mother and the infant, the causes of preterm contractions are seldom recognized before the initiation of tocolytic therapy. The administration of uterorelaxant drugs might provide time for obstetrical interventions such as screening for asymptomatic bacteriuria, antenatal

Conclusion

In the light of our results, we can conclude that the uterus-relaxing effect of terbutaline was increased notably in the presence of the PDE4 inhibitor rolipram. The combination of terbutaline + rolipram seems favorable to promote myometrium relaxation, especially in the case of genital inflammation, which condition is liable to trigger preterm birth.

Conflict of interest

No conflicts of interest.

Acknowledgement

This work was supported by the Hungarian OTKA Research Grant (K62707).

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