KKATP channels are involved in the tocolyticeffect of ß 22 agonists in pregnant rat. Norbert Lovász, Andrea Koncz, Eszter Ducza and George Falkay Department of Pharmacodynamics and Biopharmacy University of Szeged, Hungary

INTRODUCTION RESULTS Preterm birth defined as childbirth between 20 and 37 weeks of gestation by WHO, is a major determinant of neonatal mortality and morbidity and has long term adverse consequences for health. In the USA, the preterm delivery rate is 12-13%, in Europe and other developed country, reported rates are generally 5- 30 9%. The exact causes and aetiologies of preterm birth are not known. The *** incidence of preterm birth has not decreased over the years despite major 25 improvements in medical, especially perinatal care facilities and extensive medical research. In the view to decreasing the potentially maternal and foetal 20 adverse events and improving the perinatal outcome, it is a pharmacological

challenge to find new therapeutic strategies, mechanisms or combinations. In the Q R 15

clinical practice the most frequently used tocolytic agents are the ß2- 10 ** adrenoceptor (ß 2-AR) agonist (e.g. terbutaline, fenoterol, hexoprenaline,). Present study unravels the functional presence of ATP-sensitive potassium ns 5 channel (K ATP channel) and its involvement in mediating ß2-AR agonist-induced ns ns myometrial relaxation in rat myometrium at 6 and 22 days of gestation. ns ns ns ns ns 0 NP 6 8 10 12 14 15 18 20 21 22 MATERIALS AND METHODS days of pregnancy Animals: non-pregnant and 6-, 8-, 10-, 12-, 14-, 15-, 18-, 20-, 21-, 22-day pregnant SPRD rats. Quantative real-time PCR (ABI, Step One) and Western blot analysis were used to demonstrate the expressions of SUR1 gene and protein expressions in the pregnant and non-pregnant uterus, respectively. In vitro contractility study: the uterus-relaxant effect of the ß2-AR agonists (terbutaline, ritodrine and salmaterol) (10 -10-10 -5 M) were investigated on spontaneous rhythmic contractions cumulatively alone, and in the presence of Figure 1 A Changes in expression of SUR1 mRNA during pregnancy in the rat myometrium. RQ (Relative Quantity) values on different -6 KATP channel blocker glibenclamide (10 M) and KATP channel opener pinacidil days of pregnancy were compared with those in non-pregnant rats. ns: non-significant, ** denotes p< 0.01, *** p<0.001. Each bar (10 -9-10 -7 M) after 5 minutes pre-incubation. For statistical evaluations, data indicates the mean ± S.E.M, n = 5. B Representative Western blot of SUR1 protein expression in the non-pregnant (NP) and the pregnant were analysed by the ANOVA Neuman–Keuls test. rat myometrium and GAPDH as endogenous control.

6-day pregnant rat 22-day pregnant rat 4.0 ××××10 -7 2.5 ××××10 -8 ns 3.0 ××××10 -7 ns ns -8 -8 -8 100 2.0 ××××10 3.0 ××××10 ns 100 1.5 ××××10 100 (M) 2.0 ××××10 -7 1.5 ××××10 -8 100 50

*** (M) EC 50 -8 -8 1.0 ××××10 -8 1.0 ××××10 2.0 ××××10 1.0 ××××10 -7 80 EC ** (M) 80 (M) 5.0 ××××10 -9 *** 80 5 0 5 0 *** 80 0 T T+P9 T+P8 T+P7 EC 5.0 ××××10 -9 0 EC 1.0 ××××10 -8 T T+P9 T+P8 T+P7 60 60 60 60 0 0 T T+G T T+G

40 40 40 contraction(%) 40 contraction (%) contraction contraction (%) contraction(%) 20 20 20 20

0 0 0 0 -12 -11 -10 -9 -8 -7 -6 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 log [terbutaline] -11 -10 -9 -8 -7 -6 -5 log [terbutaline] log [terbutaline] log [terbutaline] 6.0 ××××10 -7 8.0 ××××10 -7 ns ns 8.0 ××10 -8 -7 ×× -8 4.0 ××××10 1.5 ××××10 6.0 ××××10 -7

* (M) ns 6.0 ××××10 -8 50 100 (M) 4.0 10 -7 -8 ×××× 100 EC -7 ns 100 100 1.0 ××××10 5 0 2.0 ××××10 (M) (M) 4.0 ××××10 -8 EC 50 50 * 2.0 ××××10 -7

EC * EC 5.0 ××××10 -9 * 80 0 80 2.0 ××××10 -8 80 80 S S+P9 S+P8 S+P7 0 S S+G 0 0 60 S S+G 60 S S+P9 S+P8 S+P7 60 60

40 40 40 40 contraction (%) contraction contraction (%) contraction contraction (%) contraction (%) contraction 20 20 20 20

0 0 0 0 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 log [salmeterol] log [salmeterol] log [salmeterol] log [salmeterol] 4.0 ××××10 -7 ns ns 4.0 ××××10 -7 4.0 ××××10 -7 ns 3.0 ××××10 -7 ** -7 3.0 ××10 -7 3.0 ××××10 -8 ×× ns 1.0 ××××10 (M) 2.0 ××××10 -7 100 50 (M)

(M) -7 100 -9 2.0 ××××10 EC 2.0 ××××10 -7 8.0 ××××10 50 50 100 100 1.0 ××××10 -7 EC

EC -9 6.0 ××××10 1.0 ××××10 -7 1.0 ××××10 -7 80 (M) 50 * 0 80 -9 4.0 ××××10 * * 80 R R+P9 R+P8 R+P7 EC 0 80 0 R R+G R R+G 2.0 ××××10 -9 60 60 0 60 60 R R+P9 R+P8 R+P7 40 40 40 40 contraction (%) contraction contraction (%) contraction contraction (%) contraction contraction (%) contraction 20 20 20 20

0 0 0 0 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 log [ritodrine] log [ritodrine] log [ritodrine] log [ritodrine]

Figure 2 Uterus-relaxing effect of the ß2-AR agonists on spontaneous rhythmic contractions in the 6-day-pregnant rat Figure 3 Uterus-relaxing effect of the ß2-AR agonists on spontaneous rhythmic contractions in the 22-day-pregnant rat myometrium (green), reversal by glibenclamide (10 -6 M) (red) and in the presence of pinacidil; 10 -9M (orange), 10 -8M (blue) myometrium (green), reversal by glibenclamide (10 -6 M) (red) and in the presence of pinacidil; 10 -9M (orange), 10 -8M -7 -7 and 10 M (pink). Inserts: changes in IC 50 values of the ß2-AR agonists on spontaneous rhythmic contractions in the 6-day- (blue) and 10 M (pink). Inserts: changes in IC 50 values of the ß2-AR agonists on spontaneous rhythmic contractions in the pregnant rat. * p < 0.05, ** p < 0.01 and *** p < 0.001. Each value denotes the mean ± S.E.M, n = 6. 22-day-pregnant rat, ns: non significant. Each value denotes the mean ± S.E.M, n = 6. CONCLUSIONS

Results of our study evidently suggest the functional presence of K ATP channel in the pregnant rat myometrium and its role in ß2-adrenoceptor agonists induced myometrial relaxation at early stage of pregnancy.

KATP channels are not involved in the tocolytic effect of the ß2-adrenoceptor agonists at term. According to the clinical observation the tocolytic effect of the ß-mimetics decrease at term. The reason of this phenomenon can be explained with the down-regulated K ATP channels near delivery.
Based on these findings, it may not be suggest that the therapeutic application of both the ß2-adrenoceptor agonist and specific K ATP channel opener as promising tocolytic agent. However, it may be used for treatment of habitual abortion.

contactcontact::lovasznorbertlovasznorbert@@pharm.upharm.u--szeged.huszeged.hu The presentation is supported by the European Union and coco--fundedfunded by the European Social Fund. “Broadening the knowledge base and supporting the long term professional sustainability of the Research University Centre of ExExcellencecellence at the University of Szeged by ensuringensuring the risingrising generation of excellent scientists.” Project number: TÁMOPTÁMOP--4.2.2/B4.2.2/B--10/110/1--20102010--00120012

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