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Relaxant effect of proton pump inhibitors on in vitro myometrium from pregnant women
European Journal of Pharmaceutical Sciences 52 (2014) 125–131
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Relaxant effect of proton pump inhibitors on in vitro myometrium from pregnant women C. Terranova a, C. Petrella b,⇑, G. Improta b, C. Severini c, F. Signore d, P. Damiani d, F. Plotti a, C. Scarpignato e, R. Angioli a a
Department of Obstetrics and Gynaecology, ‘‘Campus Bio Medico’’ University, Rome, Italy Department of Physiology and Pharmacology ‘‘V. Erspamer’’, Sapienza University, Rome, Italy Institute of Cellular Biology and Neurobiology, CNR, Rome, Italy d Department of Obstetrics and Gynaecology, San Camillo – Forlanini Hospital, Rome, Italy e Division of Gastroenterology, Department of Clinical Sciences, University of Parma, Italy b c
a r t i c l e
i n f o
Article history: Received 9 July 2013 Received in revised form 30 September 2013 Accepted 15 October 2013 Available online 9 November 2013 Keywords: Myometrial contraction Preterm delivery PPIs Tocolytic effect
a b s t r a c t Aim: In this study we investigate in in vitro myometrial tissue samples of pregnant women: (a) the effects of proton pomp inhibitors (PPIs) (omeprazole, esomeprazole, pantoprazole, lansoprazole and rabeprazole) on spontaneous contractions; (b) the muscle-relaxant efficacy of the most active PPI considered (pantoprazole) in comparison with that of other known tocolytics (nifedipine, atosiban, MgSO4, isoxsuprine); (c) the effect of pantoprazole on contractions induced by calcium (Ca++), KCl, oxytocin and prostaglandin (PGE2); (d) the possible mediators of pantoprazole relaxant effect. Methods: Organ bath studies were performed on myometrial tissue samples (40 10 10 mm) from pregnant women (38–42 weeks of gestational age) undergoing elective caesarian section. Results: All the PPIs studied reduce the spontaneous contraction of the myometrial smooth muscle. Pantoprazole is the most effective and most potent inhibitor among those analyzed. Pantoprazole also reduces the contractions induced by Ca++, KCl, oxytocin and PGE2. Neither NO, nor PGs, or the activation of Ca++-dependent K+ currents mediate the muscle-relaxant effect of this PPI. Conclusion: These data, together with the fact that PPIs almost do not present side effects, suggest that these drugs can offer new therapeutic strategies for preterm delivery. Undoubtedly, further investigations and clinical studies are necessary before adding PPIs to the list of drugs available for the treatment of preterm delivery. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The proton-pump inhibitors (PPIs) demonstrated to be potent and effective against gastric acid secretion, becoming first-choice drugs in clinical practice for the treatment of acid-peptic diseases, thanks to their significant effectiveness and excellent tolerability. The PPIs action site is the H+/K+ATPase enzyme, a family of isoenzymes present in the stomach as well as in colon, bladder, kidney, vascular smooth muscle, in airways and corpora cavernosa, and in polymorphonuclear leukocytes (van Driel and Callaghan, 1995; Marrelli et al., 1997). H+/K+ATPase is also expressed at uterine level and, in particular, among the intermediate and superficial myometrial layers. The myometrial H+/K+ATPase may be responsible for the K+ gradient maintenance through the plasmatic membrane
⇑ Corresponding author. Address: Department of Physiology and Pharmacology ‘‘V. Erspamer’’, Sapienza University of Rome, P.le Aldo Moro, 5, 00185 Rome, Italy. Tel./fax: +39 0649912487. E-mail address: [email protected] (C. Petrella). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.10.007
and for controlling the cell excitability potential (Jaisser et al., 1993). Preterm delivery, together with congenital birth defects, is the most common cause of perinatal morbidity and mortality (Gynecologist, 2003), and requires a prompt and proper therapeutic option through the administration of tocolytic drugs. So far, the main classes of drugs used as tocolytics are: b-adrenergic receptor agonists, prostaglandin (PG) synthesis inhibitors (Zuckerman et al., 1974), calcium (Ca++)-antagonists (Ulmsten et al., 1980; Sorkin et al., 1985), oxytocin antagonists (Romero et al., 2000; Group, 2001), nitric oxide (NO) donors (Di Renzo and Roura, 2006) and MgSO4 (Gordon and Iams, 1995; Taber et al., 2002). Tocolytic drugs are the cornerstone of primary pharmacologic management of preterm labor. They are intended to stop uterine contractions during a current episode of preterm labor or maintain uterine quiescence after an acute episode (Di Renzo and Roura, 2006). Nevertheless to date, there are no certainties about the capacity of tocolytic drugs to prolong the pregnancy thus improving the neonatal outcome. All the classes of tocolytic drugs have a different
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level of effectiveness and present important and not negligible side effect (Michalak et al., 1983; Caritis et al., 1990; Impey, 1993; Scudiero et al., 2000; Di Renzo and Roura, 2006). In the light of the above, the search for new tocolytic drugs to be administered in case of preterm delivery represents an unlimited area of investigation. It has been recently pointed out that omeprazole, one of the most commonly administered PPIs, reduces the spontaneous myometrial contractions and those induced by Ca++ in in vitro preparations of myometrial samples of pregnant women (Yildirim et al., 2001). Such a result, as well as the fact that the PPIs administration during pregnancy does not provoke neither an increase in fetal malformations, both severe and mild, nor an increase in neonatal complications (Lalkin et al., 1998), certainly arouse an interest about the use of PPIs in the preterm delivery therapy. Therefore, the aims of this study were: (a) To confirm the muscle-relaxant efficacy of omeprazole, and to compare it with that of other PPIs on the spontaneous contractions of in vitro myometrial tissue samples belonging to women between the 38th and the 42nd week of pregnancy. (b) To compare, on the same in vitro preparations the musclerelaxant efficacy of the PPI considered the most active, with the one of some of the most commonly used tocolytics. (c) To assess, subsequently, the muscle-relaxant action of the PPI, resulted to be the most effective on spontaneous uterine contractions, also on contractions induced by Ca++, KCl, oxytocin and prostaglandins (PGE2). (d) To investigate the possible interaction between the most effective PPI and other endogenous systems modulating contraction/relaxation in this tissue. 2. Materials and methods 2.1. Myometrium preparations Between October 2010 and June 2011, samples of myometrium were taken from enrolled term pregnant women (38–42 weeks of gestational age) undergoing to elective cesarean section, at the San Camillo-Forlanini Hospital, Rome, Italy, with the indication being one of the following: previous caesarian section or fetal malpresentation. Multiple pregnancies, patients affected by any kind of pathology related or not to the pregnancy and submitted to medical treatments were excluded. The study has been approved by the local ethics committee (San Camillo-Forlanini Hospital, Rome, Italy) and an informed written consensus was obtained from each patient. Myometrial tissue samples (40 10 10 mm) were dissected from the central portion of the hysterotomy upper margin carried out during elective caesarian section. The biopsies obtained were immediately placed in a modified physiological solution (CaCl2 1.6 mM, NaCl 119 mM, NaHCO3 25 mM, glucose 5.5 mM, KH2PO4 1.18 mM, MgSO4 1.17 mM, KCl 4.7 mM) at pH 7.4, preserved at a temperature of 4 °C and used within 24 h from the sample collection. 2.2. Measurement of the myometrium contractile activity The myometrial tissue samples were dissected in small longitudinal strips (approximately 30 5 mm), following the muscle orientation and then vertically placed in a bath for isolated organs (4000-Basile, Milano) containing the above indicated modified physiological solution and oxygenated with a solution consisting of 95% O2 and 5% CO2 at 37 °C.
The tissue strips were subjected to a constant tension of 1 g and left to balance for 60 min before starting the test of the substances under study. The tissue tension was isometrically measured by means of a strain-gauge transducer (DY 1, Basile, Milan, Italy) and digitally recorded by means of 17304 – NEW DataCapsuleEvo Digital Recorder (Basile, Milan, Italy). After the equilibration period, concentration–response curves were obtained by adding cumulative concentrations of the different drugs at 30 min intervals. Three strips from three donors were used for each compounds tested. 2.3. Effect of PPIs on spontaneous uterine contractile activity In a first series of experiments, the effect on the spontaneous contractile activity of uterine tissue of subsequent and increasing concentrations (from 100 lM up to 1000 lM) of five different PPIs (omeprazole, esomeprazole, pantoprazole, lansoprazole and rabeprazole) was recorded. Then, the relative potency of the most effective PPI, pantoprazole, was compared to the potency of other reference tocolytics: isoxsuprine (25–500 lM), atosiban (25–150 lM), nifedipine (1– 10 lM) and MgSO4 (1000–2000 lM). 2.4. Effect of pantoprazole on uterine contractions induced by KCl, oxytocin, PGE2 and Ca++ In a second series of experiments, the muscle-relaxant effect of the PPI, turning out be the most potent and effective, pantoprazole, on uterine contractions induced by KCl (80 mM), oxytocin (1 lM), PGE2 (1 lM) and Ca++ (8 mM), was assessed (three strips from the same donor were analyzed at the same time, within 24 and 48 h from the dissection, using three donors for each compound). After 10–40 min of incubation with the contractile agents, a single concentration of pantoprazole (from 100 to1000 lM) was added and left for about 20 min. 2.5. Effect of the pre-treatment with indomethacin, L-NAME, apamin and iberiotoxin on pantoprazole tocolytic activity Finally, in a third group of myometrial tissue samples, the pantoprazole (500 lM) relaxant efficacy was measured after 15 min pre-treatment with a PG synthesis inhibitor, indomethacin (3 lM), with NO synthesis inhibitor, L-NAME (30 lM), with Ca++-dependent K+ channel blockers, apamin (100 nM) and iberiotoxin (100 nM), in order to identify the possible interaction with other endogenous systems modulating contraction/relaxation in this tissue. 2.6. Drugs All the PPIs tested were purchased by Sigma–Aldrich, Italy (omeprazole: cod. O104; esomeprazole: cod. E7906; pantoprazole: cod. P0021; lansoprazole: cod. L8533; rabeprazole: cod. SML0476). Nifedipine (cod. 1075), oxytocin (cod. 1910), PGE2 (cod. 2296), L-NAME (cod. 0665) were purchased by Tocris, United Kingdom. Indomethacin was purchased by Promedica, Parma, Italy (Liometacen), Apamin (cod. L8407) and iberiotoxin (cod. L8211) were bought by Latoxan (France). Finally, isoxsuprine (cod. 579-56-6) was purchased by Santa Cruz Biotechnology, Germany. Apamin, iberiotoxin, isoxsuprine, PGE2 and oxytocin were dissolved in phosphate buffered saline (PBS). All the other compounds were dissolved in a dimethyl sulfoxide (DMSO) stock solution and then diluted in PBS, to obtain the test concentrations, never exceeding 0.1% of DMSO, to avoid possible influence on the spontaneous contractile activity of the myometrium.
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2.7. Statistical analysis To assess the efficacy of the drugs under consideration, for each concentration used, a percentage of contractions compared to the extent of the spontaneous contractions was calculated, quantifying the amplitude of any contraction. The relaxant effect induced by pantoprazole in the pre-contracted tissues was calculated associating the value of 100 to the maximum contraction obtained with the agonists used. The data are presented as mean values ± Standard Error (SE) and analyzed using the ANOVA test for repeated measurements followed by the Bonferroni test to evaluate the significance. A value of P < 0.05 was considered significant. All the results were then interpolated creating the proper concentration–response curve, where the concentration is expressed as log to identify the IC50, the concentration capable of inhibiting the effect observed by 50% and, therefore, to compare the potency of the drugs under consideration.
3. Results All the PPIs studied (omeprazole, esomeprazole, pantoprazole, lansoprazole and rabeprazole), in a range of concentrations between 100 and 1000 lM, induced a concentration-dependent decrease of the spontaneous contractile activity of isolated myometrial smooth muscle preparations of women between the 38th and 42nd week of pregnancy (Fig. 1). The most effective among the PPIs was pantoprazole that, at the concentration of 250 lM, significantly inhibits the spontaneous contractility by approximately 50% and, at 1000 lM concentration, relaxes the myometrium completely. The IC50 values (the concentration producing an inhibitory effect by 50%) concerning PPI muscle-relaxing effect, calculated by linear regression from cumulative concentration–response curves, are reported in Table 1, and show that the pantoprazole IC50 value significantly differs from those of the other PPIs considered. Therefore, for the five PPIs the order of potency is the following: pantoprazole > rabeprazole > lansoprazole > esomeprazole = omeprazole. In particular, pantoprazole besides having a potency significantly higher than the other four PPIs, at the maximum concentration used (1000 lM), has a greater efficacy because being capable of reducing the amplitude of contractions almost completely, as indicated by the % relaxation index, HC (Table 1). All the PPIs studied do not change the spontaneous contraction frequency, except for the pantoprazole at the maximum (500 lM)
Fig. 1. The cumulative concentration–response curves of different PPIS on the spontaneous contractile activity of myometrium from pregnant women (38–42nd week). Each point represents the mean ± SE of 3 independent experiments in triplicate; ⁄ and ⁄⁄ indicate p < 0.05 and p < 0.01, respectively.
Table 1 Relaxant effect of different tocolytics on spontaneous myometrial contraction in pregnant women (between 38th and 42nd week). The IC 50 values are calculated by linear regression from cumulative concentration–response curves. HC is the highest concentration tested producing the % of relaxation indicated. Drugs
*
IC50–log (range)
% Relaxation (HC)
PPIs
PANTOPRAZOLE OMEPRAZOLE ESOMEPRAZOLE LANSOPRAZOLE RABEPRAZOLE
4.09 ( 3.24 ( 3.28 ( 3.40 ( 3.51 (
4.19/ 3.37/ 3.43/ 3.56/ 3.66/
3.98) 3.09)* 3.13)* 3.25)* 3.37)*
94 ± 5.6 45 ± 4.8 48 ± 3.5 45 ± 6.8 46 ± 3.8
(1 mM) (1 mM)* (1 mM)* (1 mM)* (1 mM)*
TOCOLYTICS
ISOXSUPRINE ATOSIBAN NIFEDIPINE MgSO4
4.05 ( 4.26 ( 6.15 ( 2.11 (
4.16/ 4.35/ 6.37/ 2.28/
3.94) 4.17) 5.92)* 1.93)*
86 ± 7.5 (0.4 mM) 93 ± 6.8 (0.15 mM) 92 ± 7.5 (10 lM) 90 ± 4.9 (2.5 mM)
p < 0.05, versus pantoprazole.
concentration used, that decreased the number of spikes in the trace (Fig. 2). The concentration–response curves relative to the % spontaneous contractility induced by pantoprazole, compared to the effect of other tocolytics (nifedipine, atosiban, isoxsuprine, MgSO4), show that the relaxing effect induced by nifedipine is reached at the lowest molar concentrations with respect to those of the other tocolytics (Fig. 3). Concentrations with the same order of magnitude are necessary for the tocolytic action induced by isoxsuprine and atosiban. Follows the pantoprazole, that, compared to the previous tocolytics, is from two to five times less potent, and finally MgSO4 that to induce a myometrium relaxation requires 8 times higher molar concentrations compared to pantoprazole. The IC50 values calculated for each agent confirm these data (Table 1) and indicate that their relative potency is the following: nifedipine o atosiban > isoxsuprine > pantoprazole o MgSO4. In a second phase of this study, by testing the pantoprazole action on myometrium sample pre-contracted by oxytocin (1 lM), PGE2 (1 lM), KCl (80 mM) and Ca++ (8 mM), it has been shown that pantoprazole at 250, 500 and 1000 lM concentration inhibits, significantly and in concentration–response manner, the contractions induced by each of the agents used (Fig. 4). The concentration of pantoprazole necessary to reduce the contractile activity of the four agents does not significantly differ, being approximately 500 lM (Fig. 4 and Table 2). Thus, this concentration of pantoprazole was chosen in the subsequent experiments of pre-treatment with antagonists. The pre-treatment with indomethacin (3 lM), L-NAME (30 lM), apamin (100 nM) and iberiotoxin (100 nM) does not modify the pantoprazole (500 lM) muscle-relaxant effect on contractions induced by KCl, oxytocin, PGE2, and Ca++, as reported in Table 2.
Fig. 2. Representative recording showing the effect of pantoprazole (500 lM) on spontaneous contractions of the myometrium of pregnant women (38–42nd week).
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Fig. 3. The cumulative concentration–response curves of known tocolytic drugs on the spontaneous contractile activity of myometrium from pregnant women (38– 42nd week) in comparison with pantoprazole. Each point represents the mean ± SE of 3 independent experiments in triplicate; ⁄ and ⁄⁄ indicate p < 0.05 and p < 0.01, respectively.
4. Discussion In the present study we demonstrate that PPIs reduce in a concentration manner the spontaneous contraction of the myometrial smooth muscle of women between 38th and 42nd week of
pregnancy, confirming that such class of drugs may play a role in the basal control of the uterine muscle tone in vitro. Pantoprazole demonstrated to be the most potent and most effective relaxant agent among the PPIs analyzed (omeprazole, esomeprazole, rabeprazole and lansoprazole). The potency order of pantoprazole muscle relaxing effect, in comparison with the other known tocolytics, is the following: significantly lower than nifedipine, approximately of the same order of magnitude as oxytocin antagonist, atosiban, and b2-agonist, isoxsuprine, but significantly higher compared to MgSO4. Pantoprazole can also reduce the contractions induced by Ca++, by the depolarizing agent KCl and by the two physiological agents of uterine contractions, oxytocin and PGE2. Furthermore, as for the possible interaction with other mechanisms underlying the relaxant effect of the PPI, our data show that the pantoprazole relaxant effect is not affected by indomethacin, the COX inhibitor, by L-NAME, the NO synthesis inhibitor, by apamin and iberiotoxin, the Ca++-dependent K+ channel blockers. Therefore, neither NO, nor PGs, or the activation of Ca++-dependent K+ currents mediate the effect of this PPI-induced decrease of myometrial tone. It is well known that PPIs inhibit the H+/K+ ATPase by a covalent modification of the enzyme (Shamburek and Schubert, 1993). H+/K+ATPase isoforms are present in organs other than the stomach including the vascular smooth muscle cells. Omeprazole, the
Fig. 4. Diagrams of miolytic effect of different concentrations of pantoprazole on contractions induced by oxytocin 1 lM (panel A), PGE2 1 lM (panel B), KCl 80 mM (panel C) and Ca++ 8 mM (panel D) in the myometrium of pregnant women (38–42nd week). Each point represents the mean ± SE of 3 independent experiments in triplicate; ⁄⁄ indicate p < 0.01. Representative recordings of effect of different pantoprazole concentrations were reported.
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Table 2 Effect of pantoprazole (500 lM) on the myometrium contraction induced by KCl, oxytocin, PGE2 and Ca++ in uterine samples of women during pregnancy (between 38th and 42nd week), in the absence and presence of different inhibitors (L-NAME, indomethacin, iberiotoxin, apamin). Drugs
% contraction induced by: KCl (80 mM)
Vehicle PANTOPRAZOLE (500 lM)
*
+Saline +L-NAME (30 lM) +INDOMETHACIN (3 lM) +IBERIOTOXIN (100 nM) +APAMIN (100 nM)
100 ± 10 38 ± 10 42 ± 12 36 ± 6 34 ± 9 40 ± 11
Oxytocin (1 lM) *
100 ± 15 32 ± 8 36 ± 8 29 ± 8 31 ± 4 38 ± 8
PGE2 (1 lM) 100 ± 12 26 ± 12 32 ± 12 25 ± 9 34 ± 10 29 ± 12
*
Ca++ (8 mM) 100 ± 9* 26 ± 9 27 ± 6 20 ± 10 19 ± 8 29 ± 10
* Indicates p < 0.01 by comparing the maximum effect (100%) induced in controls (vehicle) by the contractile agents with the one induced by the other pharmacological treatments.
H+/K+ATPase inhibitor, relaxes the spontaneous muscular tone of the experimental animal’s trachea (Rhoden et al., 1996). Furthermore, a benzimidazolic derivative (NC-1300) reduces the contractions induced by the acetylcholine in the guinea pig ileum (Okabe et al., 1996), suggesting that such derivatives can reduce the smooth muscle tone. In addition, a single work (Yildirim et al., 2001) reports that omeprazole inhibits the uterine contraction, and that the PPI relaxing effect is not mediated nor by the products resulting from the degradation in the COX nor by NO, but consequent to Ca++ channels blockers. Therefore the results of our study, on one hand confirm the data reported by Yildirim (Yildirim et al., 2001), but also extend the knowledge about such issue offering new elements for the discussion. KCl is a depolarizing agent that, by releasing acetylcholine, induces smooth muscle contraction by means of a tension-dependent intercellular Ca++ flow (Karaki, 1987). The periodic contractions of human myometrium are evoked by the release of Ca++ from intracellular deposits and by the flow through tension dependant L-type Ca++ channels (Sanborn, 2000; Wray et al., 2003). The facts that pantoprazole is capable of reducing uterine contractions induced by KCl is suggestive of an inhibiting interaction with cholinergic neuronal mechanisms, confirmed by the consequent muscle-relaxing effects in other above reported tissues (Okabe et al., 1996), and it also responsible for the intercellular Ca++ reduction. Pantoprazole relaxes the myometrium contracted by oxytocin. The contractions induced by oxytocin are due to a chemical– mechanical combination consisting in the activation of receptors associated to the phospholipase C, inducing the production of IP3 and the release of Ca++ from intracellular deposits (Giraldi et al., 1990). Furthermore, oxytocin increases the contracting system sensitivity to Ca++ signals through the rho-kinase (Bradley et al., 1998), essential for the efficacy of the combination between an increase in Ca++ and the tension developed. Once again an interference with Ca++ intracellular levels emerges among the possible targets of the PPIs muscle-relaxant action on oxytocin-induced contractions. Pantoprazole significantly reduces uterine contractions induced by PGE2. PGE2 are known as potent oxytocic agents and their action is associated to the activation of intracellular sites releasing Ca++ (Egarter and Husslein, 1992). In the myometrium of ‘‘preterm’’ and ‘‘at term’’ women in labor the COX is expressed. COX inhibitors reduce the PG production in the myometrium and are considered tocolytics (Tao et al., 2005; Choi et al., 2007). In any case it was assumed that non steroid anti-inflammatory drugs manifest their muscle relaxing effect by means of different mechanisms resulting from PG synthesis inhibition, for example by means of intercellular Ca++ reduction. On this subject, our results have shown that indomethacin, at concentrations capable of inhibiting the PG synthesis in the myometrium, does not modify the pantoprazole tocolytic action. Such datum if, on one hand, demonstrates that prostaglandinergic
pathways, and therefore the COX inhibition, do not seem to be involved in the pantoprazole tocolytic action, on the other hand, indirectly, confirms the existence of another main mediator of the contracting action by PGE2 which represents the target of the pantoprazole muscle-relaxing action on contractions induced by the PGE2, that we assume could be the intracellular Ca++. Also NO pathways are capable of relaxing animal and human myometrium (Bradley et al., 1998) but, as recently demonstrated (Buxton et al., 2001), not through the cGMP activation, responsible for mediating most of the actions of this new neurotransmission system, but rather by means of the direct or indirect activation of K+ channels through Ca++. Since the relaxation induced by NO donors can be prevented by some toxins, specific inhibitors of these high conductance channels for K+, it is highly probable that one more of these channels, as expressed in the human preterm myometrium, can be the target for NO and other tocolytics. The mechanism regulating the muscle-relaxant action would consist in an increase in intracellular Ca++, responsible for the K+ channel activation associated to Ca++, whose opening would induce the activation of tension-dependent channels permeable to K+. It could provoke cell hyperpolarization and consequently relaxation. Actually such conceivable mechanism has not been demonstrated to take place in myometrium. In any case, about this issue, the results, we obtained, demonstrate that in the muscle relaxing action induced by pantoprazole, neither NO nor K+ channels activated by Ca++ play the role of mediators. The pre-treatment of myometrium preparation, both with NO synthesis inhibitor or K+ channel blockers, apamin and iberiotoxin, does not modify the smooth muscle-relaxing effect of this PPI on oxytocin- induced contractions. All these considerations suggest that PPIs induce a smooth muscle relaxing effect on the gravid uterus probably using a target common to other systems and to different endogenous mechanisms capable of controlling the contracting and/or relaxing capacity of the uterine smooth muscular fibers. Most of the tocolytic agents regulate the intracellular Ca++ concentration although the function of such channels during delivery is still unknown. The beginning of delivery is associated to many myometrial modification including alterations in electrophysiological mechanisms (gap junctions, membrane ion channels) (Miller et al., 1989; Garfield et al., 1992) and biochemical processes (actin/myosin coupling, receptors) controlling myometrium contractility and relaxation (Egarter and Husslein, 1992; Hertelendy and Zakar, 2004). For example, it has been recently observed that in the myometrial cells of gravid rats during the last period of gestation the expression of rapid Na+ channels increases more than the expression of slow Ca++ channels (Ohya and Sperelakis, 1989; Sperelakis et al., 1992). Also K+ channels play a role in the regulation of uterine contractility, and modifications in the expression and in the function of such channels can be responsible for myometrial modifications during the different phases of the pregnancy (Lundgren et al., 1997; Knock et al., 1999; Brainard et al., 2005).
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The fact that pantoprazole has a relaxing effect on uterine contractions induced by different contracting agents but all using Ca++ as final mediator of their action, let us assume, by deduction, that PPIs may interfere with the intracellular concentration of this ion by reducing it even if we are not able to define the different phases leading to the intracellular Ca++ reduction. Nevertheless, we cannot exclude that other signal mechanisms regulating smooth muscle contraction may intervene in the pantoprazole-induced inhibition. Furthermore, the data of this study do not permit to define whether the PPIs action is specific or non specific. It is important to remark that the PPIs receptor site of action, the H+/K+ ATPase, has a high level of structural homology with Na+/K+ ATPases in certain tissue such as colon, presenting a similar pharmacologic profile as both are sensitive to ouabain. Such ATPases can transport Na+ outside the cell exchanging it with K+ due to its sensitivity to cations. It has been recently demonstrated (Veklich et al., 2007) that omeprazole and lansoprazole (10–100 lM) inhibit also the activity of such pump at plasma membrane level in myometrial smooth muscle cells with a consequent reduction in Na+/K+ exchanges, an increase in intracellular K+ and a reduction in cell excitability. However, the lack of data clearly indicating the function of both proton pumps in human myometrium does not allow us to accurately define the mechanism regulating the muscle relaxant action in PPIs. 5. Conclusions To conclude, this study clearly confirms the relaxant effect of PPIs in human myometrium in vitro. All the PPIs, tested at sufficient concentration, are capable of relaxing the myometrial tissue samples even if their potency compared to other well known tocolytics is almost always lower. Notwithstanding, the pharmacological potency relative to the muscle relaxant action tested in vitro does not correspond to a clinic tocolytic efficacy, but it merely indicates that a certain drug is more effective at a lower dose. The extrapolation of the concentration in a bath for isolated tissues at plasmatic level in patients is full of uncertainties. First of all, it has been demonstrated that the plasmatic levels of some of this tocolytic agents widely vary due to differences in their pharmacokinetics and they are not always correlated with their muscle relaxant action. Secondly, similar concentrations in vitro and in the patients’ plasma in vivo do not correspond to similar concentrations in the tissues. Usually, clinical studies on different tocolytics demonstrated that the concentrations in vitro were higher than plasmatic concentrations in vivo. Such discrepancy could be the cause of the ineffectiveness of some tocolytics in preventing preterm delivery. Furthermore, in the clinical practice, toxicity is a limit for the plasmatic concentrations to be reached. Most of the time-concentrations producing a muscle relaxing effect can be associated to severe side effects. As the PPIs almost do not induce side effects means that such drugs can offer new therapeutic strategies even if, undoubtedly, further investigations to understand the mechanism regulating their tocolytic action and clinical studies in vivo are necessary before adding PPIs to the list of drugs available for the treatment of preterm delivery. References Bradley, K.K., Buxton, I.L., Barber, J.E., McGaw, T., Bradley, M.E., 1998. Nitric oxide relaxes human myometrium by a cGMP-independent mechanism. Am. J. Physiol. 275, C1668–C1673. Brainard, A.M., Miller, A.J., Martens, J.R., England, S.K., 2005. Maxi-K channels localize to caveolae in human myometrium: a role for an actin-channelcaveolin complex in the regulation of myometrial smooth muscle K+ current. Am. J. Physiol. Cell Physiol. 289, C49–C57. Buxton, I.L., Kaiser, R.A., Malmquist, N.A., Tichenor, S., 2001. NO-induced relaxation of labouring and non-labouring human myometrium is not mediated by cyclic GMP. Br. J. Pharmacol. 134, 206–214.
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Marrelli, S.P., Zhao, X., Allen, J.C., 1997. Molecular evidence for a vascular smooth muscle H+-K+-ATPase. Am. J. Physiol. 272, H869–H874. Michalak, D., Klein, V., Marquette, G.P., 1983. Myocardial ischemia: a complication of ritodrine tocolysis. Am. J. Obstet. Gynecol. 146, 861–862. Miller, S.M., Garfield, R.E., Daniel, E.E., 1989. Improved propagation in myometrium associated with gap junctions during parturition. Am. J. Physiol. 256, C130– C141. Ohya, Y., Sperelakis, N., 1989. Fast Na+ and slow Ca2+ channels in single uterine muscle cells from pregnant rats. Am. J. Physiol. 257, C408–C412. Okabe, S., Amagase, K., Fujita, H., Iwata, K., Satake, N., Shibata, S., 1996. Vasoinhibitory effect of leminoprazole, a H+, K(+)-ATPase inhibitor, on rat aortic rings. Gen. Pharmacol. 27, 117–121. Rhoden, K.J., Tallini, G., Douglas, J.S., 1996. H+-K+ ATPase inhibitors cause relaxation of guinea pig and human airway smooth muscle in vitro. J. Pharmacol. Exp. Ther. 276, 897–903. Romero, R., Sibai, B.M., Sanchez-Ramos, L., Valenzuela, G.J., Veille, J.C., Tabor, B., Perry, K.G., Varner, M., Goodwin, T.M., Lane, R., Smith, J., Shangold, G., Creasy, G.W., 2000. An oxytocin receptor antagonist (atosiban) in the treatment of preterm labor: a randomized, double-blind, placebo-controlled trial with tocolytic rescue. Am. J. Obstet. Gynecol. 182, 1173–1183. Sanborn, B.M., 2000. Relationship of ion channel activity to control of myometrial calcium. J. Soc. Gynecol. Investig. 7, 4–11. Scudiero, R., Khoshnood, B., Pryde, P.G., Lee, K.S., Wall, S., Mittendorf, R., 2000. Perinatal death and tocolytic magnesium sulfate. Obstet. Gynecol. 96, 178–182. Shamburek, R.D., Schubert, M.L., 1993. Pharmacology of gastric acid inhibition. Baillieres Clin. Gastroenterol. 7, 23–54. Sorkin, E.M., Clissold, S.P., Brogden, R.N., 1985. Nifedipine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy, in ischaemic heart disease, hypertension and related cardiovascular disorders. Drugs 30, 182–274. Sperelakis, N., Inoue, Y., Ohya, Y., 1992. Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus. Mol. Cell. Biochem. 114, 79–89. Taber, E.B., Tan, L., Chao, C.R., Beall, M.H., Ross, M.G., 2002. Pharmacokinetics of ionized versus total magnesium in subjects with preterm labor and preeclampsia. Am. J. Obstet. Gynecol. 186, 1017–1021. Tao, D.L., Xu, Y.Z., Huang, Y., Wei, F., Wu, J.G., Xu, D.F., 2005. The synthesis and characteristic of Co3O4 nanocrystals. Guang Pu Xue Yu Guang Pu Fen Xi 25, 5–9. Ulmsten, U., Andersson, K.E., Wingerup, L., 1980. Treatment of premature labor with the calcium antagonist nifedipine. Arch. Gynecol. 229, 1–5. van Driel, I.R., Callaghan, J.M., 1995. Proton and potassium transport by H+/K(+)ATPases. Clin. Exp. Pharmacol. Physiol. 22, 952–960.
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Relaxant effect of proton pump inhibitors on in vitro myometrium from pregnant women C. Terranova a, C. Petrella b,⇑, G. Improta b, C. Severini c, F. Signore d, P. Damiani d, F. Plotti a, C. Scarpignato e, R. Angioli a a
Department of Obstetrics and Gynaecology, ‘‘Campus Bio Medico’’ University, Rome, Italy Department of Physiology and Pharmacology ‘‘V. Erspamer’’, Sapienza University, Rome, Italy Institute of Cellular Biology and Neurobiology, CNR, Rome, Italy d Department of Obstetrics and Gynaecology, San Camillo – Forlanini Hospital, Rome, Italy e Division of Gastroenterology, Department of Clinical Sciences, University of Parma, Italy b c
a r t i c l e
i n f o
Article history: Received 9 July 2013 Received in revised form 30 September 2013 Accepted 15 October 2013 Available online 9 November 2013 Keywords: Myometrial contraction Preterm delivery PPIs Tocolytic effect
a b s t r a c t Aim: In this study we investigate in in vitro myometrial tissue samples of pregnant women: (a) the effects of proton pomp inhibitors (PPIs) (omeprazole, esomeprazole, pantoprazole, lansoprazole and rabeprazole) on spontaneous contractions; (b) the muscle-relaxant efficacy of the most active PPI considered (pantoprazole) in comparison with that of other known tocolytics (nifedipine, atosiban, MgSO4, isoxsuprine); (c) the effect of pantoprazole on contractions induced by calcium (Ca++), KCl, oxytocin and prostaglandin (PGE2); (d) the possible mediators of pantoprazole relaxant effect. Methods: Organ bath studies were performed on myometrial tissue samples (40 10 10 mm) from pregnant women (38–42 weeks of gestational age) undergoing elective caesarian section. Results: All the PPIs studied reduce the spontaneous contraction of the myometrial smooth muscle. Pantoprazole is the most effective and most potent inhibitor among those analyzed. Pantoprazole also reduces the contractions induced by Ca++, KCl, oxytocin and PGE2. Neither NO, nor PGs, or the activation of Ca++-dependent K+ currents mediate the muscle-relaxant effect of this PPI. Conclusion: These data, together with the fact that PPIs almost do not present side effects, suggest that these drugs can offer new therapeutic strategies for preterm delivery. Undoubtedly, further investigations and clinical studies are necessary before adding PPIs to the list of drugs available for the treatment of preterm delivery. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The proton-pump inhibitors (PPIs) demonstrated to be potent and effective against gastric acid secretion, becoming first-choice drugs in clinical practice for the treatment of acid-peptic diseases, thanks to their significant effectiveness and excellent tolerability. The PPIs action site is the H+/K+ATPase enzyme, a family of isoenzymes present in the stomach as well as in colon, bladder, kidney, vascular smooth muscle, in airways and corpora cavernosa, and in polymorphonuclear leukocytes (van Driel and Callaghan, 1995; Marrelli et al., 1997). H+/K+ATPase is also expressed at uterine level and, in particular, among the intermediate and superficial myometrial layers. The myometrial H+/K+ATPase may be responsible for the K+ gradient maintenance through the plasmatic membrane
⇑ Corresponding author. Address: Department of Physiology and Pharmacology ‘‘V. Erspamer’’, Sapienza University of Rome, P.le Aldo Moro, 5, 00185 Rome, Italy. Tel./fax: +39 0649912487. E-mail address: [email protected] (C. Petrella). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.10.007
and for controlling the cell excitability potential (Jaisser et al., 1993). Preterm delivery, together with congenital birth defects, is the most common cause of perinatal morbidity and mortality (Gynecologist, 2003), and requires a prompt and proper therapeutic option through the administration of tocolytic drugs. So far, the main classes of drugs used as tocolytics are: b-adrenergic receptor agonists, prostaglandin (PG) synthesis inhibitors (Zuckerman et al., 1974), calcium (Ca++)-antagonists (Ulmsten et al., 1980; Sorkin et al., 1985), oxytocin antagonists (Romero et al., 2000; Group, 2001), nitric oxide (NO) donors (Di Renzo and Roura, 2006) and MgSO4 (Gordon and Iams, 1995; Taber et al., 2002). Tocolytic drugs are the cornerstone of primary pharmacologic management of preterm labor. They are intended to stop uterine contractions during a current episode of preterm labor or maintain uterine quiescence after an acute episode (Di Renzo and Roura, 2006). Nevertheless to date, there are no certainties about the capacity of tocolytic drugs to prolong the pregnancy thus improving the neonatal outcome. All the classes of tocolytic drugs have a different
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level of effectiveness and present important and not negligible side effect (Michalak et al., 1983; Caritis et al., 1990; Impey, 1993; Scudiero et al., 2000; Di Renzo and Roura, 2006). In the light of the above, the search for new tocolytic drugs to be administered in case of preterm delivery represents an unlimited area of investigation. It has been recently pointed out that omeprazole, one of the most commonly administered PPIs, reduces the spontaneous myometrial contractions and those induced by Ca++ in in vitro preparations of myometrial samples of pregnant women (Yildirim et al., 2001). Such a result, as well as the fact that the PPIs administration during pregnancy does not provoke neither an increase in fetal malformations, both severe and mild, nor an increase in neonatal complications (Lalkin et al., 1998), certainly arouse an interest about the use of PPIs in the preterm delivery therapy. Therefore, the aims of this study were: (a) To confirm the muscle-relaxant efficacy of omeprazole, and to compare it with that of other PPIs on the spontaneous contractions of in vitro myometrial tissue samples belonging to women between the 38th and the 42nd week of pregnancy. (b) To compare, on the same in vitro preparations the musclerelaxant efficacy of the PPI considered the most active, with the one of some of the most commonly used tocolytics. (c) To assess, subsequently, the muscle-relaxant action of the PPI, resulted to be the most effective on spontaneous uterine contractions, also on contractions induced by Ca++, KCl, oxytocin and prostaglandins (PGE2). (d) To investigate the possible interaction between the most effective PPI and other endogenous systems modulating contraction/relaxation in this tissue. 2. Materials and methods 2.1. Myometrium preparations Between October 2010 and June 2011, samples of myometrium were taken from enrolled term pregnant women (38–42 weeks of gestational age) undergoing to elective cesarean section, at the San Camillo-Forlanini Hospital, Rome, Italy, with the indication being one of the following: previous caesarian section or fetal malpresentation. Multiple pregnancies, patients affected by any kind of pathology related or not to the pregnancy and submitted to medical treatments were excluded. The study has been approved by the local ethics committee (San Camillo-Forlanini Hospital, Rome, Italy) and an informed written consensus was obtained from each patient. Myometrial tissue samples (40 10 10 mm) were dissected from the central portion of the hysterotomy upper margin carried out during elective caesarian section. The biopsies obtained were immediately placed in a modified physiological solution (CaCl2 1.6 mM, NaCl 119 mM, NaHCO3 25 mM, glucose 5.5 mM, KH2PO4 1.18 mM, MgSO4 1.17 mM, KCl 4.7 mM) at pH 7.4, preserved at a temperature of 4 °C and used within 24 h from the sample collection. 2.2. Measurement of the myometrium contractile activity The myometrial tissue samples were dissected in small longitudinal strips (approximately 30 5 mm), following the muscle orientation and then vertically placed in a bath for isolated organs (4000-Basile, Milano) containing the above indicated modified physiological solution and oxygenated with a solution consisting of 95% O2 and 5% CO2 at 37 °C.
The tissue strips were subjected to a constant tension of 1 g and left to balance for 60 min before starting the test of the substances under study. The tissue tension was isometrically measured by means of a strain-gauge transducer (DY 1, Basile, Milan, Italy) and digitally recorded by means of 17304 – NEW DataCapsuleEvo Digital Recorder (Basile, Milan, Italy). After the equilibration period, concentration–response curves were obtained by adding cumulative concentrations of the different drugs at 30 min intervals. Three strips from three donors were used for each compounds tested. 2.3. Effect of PPIs on spontaneous uterine contractile activity In a first series of experiments, the effect on the spontaneous contractile activity of uterine tissue of subsequent and increasing concentrations (from 100 lM up to 1000 lM) of five different PPIs (omeprazole, esomeprazole, pantoprazole, lansoprazole and rabeprazole) was recorded. Then, the relative potency of the most effective PPI, pantoprazole, was compared to the potency of other reference tocolytics: isoxsuprine (25–500 lM), atosiban (25–150 lM), nifedipine (1– 10 lM) and MgSO4 (1000–2000 lM). 2.4. Effect of pantoprazole on uterine contractions induced by KCl, oxytocin, PGE2 and Ca++ In a second series of experiments, the muscle-relaxant effect of the PPI, turning out be the most potent and effective, pantoprazole, on uterine contractions induced by KCl (80 mM), oxytocin (1 lM), PGE2 (1 lM) and Ca++ (8 mM), was assessed (three strips from the same donor were analyzed at the same time, within 24 and 48 h from the dissection, using three donors for each compound). After 10–40 min of incubation with the contractile agents, a single concentration of pantoprazole (from 100 to1000 lM) was added and left for about 20 min. 2.5. Effect of the pre-treatment with indomethacin, L-NAME, apamin and iberiotoxin on pantoprazole tocolytic activity Finally, in a third group of myometrial tissue samples, the pantoprazole (500 lM) relaxant efficacy was measured after 15 min pre-treatment with a PG synthesis inhibitor, indomethacin (3 lM), with NO synthesis inhibitor, L-NAME (30 lM), with Ca++-dependent K+ channel blockers, apamin (100 nM) and iberiotoxin (100 nM), in order to identify the possible interaction with other endogenous systems modulating contraction/relaxation in this tissue. 2.6. Drugs All the PPIs tested were purchased by Sigma–Aldrich, Italy (omeprazole: cod. O104; esomeprazole: cod. E7906; pantoprazole: cod. P0021; lansoprazole: cod. L8533; rabeprazole: cod. SML0476). Nifedipine (cod. 1075), oxytocin (cod. 1910), PGE2 (cod. 2296), L-NAME (cod. 0665) were purchased by Tocris, United Kingdom. Indomethacin was purchased by Promedica, Parma, Italy (Liometacen), Apamin (cod. L8407) and iberiotoxin (cod. L8211) were bought by Latoxan (France). Finally, isoxsuprine (cod. 579-56-6) was purchased by Santa Cruz Biotechnology, Germany. Apamin, iberiotoxin, isoxsuprine, PGE2 and oxytocin were dissolved in phosphate buffered saline (PBS). All the other compounds were dissolved in a dimethyl sulfoxide (DMSO) stock solution and then diluted in PBS, to obtain the test concentrations, never exceeding 0.1% of DMSO, to avoid possible influence on the spontaneous contractile activity of the myometrium.
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2.7. Statistical analysis To assess the efficacy of the drugs under consideration, for each concentration used, a percentage of contractions compared to the extent of the spontaneous contractions was calculated, quantifying the amplitude of any contraction. The relaxant effect induced by pantoprazole in the pre-contracted tissues was calculated associating the value of 100 to the maximum contraction obtained with the agonists used. The data are presented as mean values ± Standard Error (SE) and analyzed using the ANOVA test for repeated measurements followed by the Bonferroni test to evaluate the significance. A value of P < 0.05 was considered significant. All the results were then interpolated creating the proper concentration–response curve, where the concentration is expressed as log to identify the IC50, the concentration capable of inhibiting the effect observed by 50% and, therefore, to compare the potency of the drugs under consideration.
3. Results All the PPIs studied (omeprazole, esomeprazole, pantoprazole, lansoprazole and rabeprazole), in a range of concentrations between 100 and 1000 lM, induced a concentration-dependent decrease of the spontaneous contractile activity of isolated myometrial smooth muscle preparations of women between the 38th and 42nd week of pregnancy (Fig. 1). The most effective among the PPIs was pantoprazole that, at the concentration of 250 lM, significantly inhibits the spontaneous contractility by approximately 50% and, at 1000 lM concentration, relaxes the myometrium completely. The IC50 values (the concentration producing an inhibitory effect by 50%) concerning PPI muscle-relaxing effect, calculated by linear regression from cumulative concentration–response curves, are reported in Table 1, and show that the pantoprazole IC50 value significantly differs from those of the other PPIs considered. Therefore, for the five PPIs the order of potency is the following: pantoprazole > rabeprazole > lansoprazole > esomeprazole = omeprazole. In particular, pantoprazole besides having a potency significantly higher than the other four PPIs, at the maximum concentration used (1000 lM), has a greater efficacy because being capable of reducing the amplitude of contractions almost completely, as indicated by the % relaxation index, HC (Table 1). All the PPIs studied do not change the spontaneous contraction frequency, except for the pantoprazole at the maximum (500 lM)
Fig. 1. The cumulative concentration–response curves of different PPIS on the spontaneous contractile activity of myometrium from pregnant women (38–42nd week). Each point represents the mean ± SE of 3 independent experiments in triplicate; ⁄ and ⁄⁄ indicate p < 0.05 and p < 0.01, respectively.
Table 1 Relaxant effect of different tocolytics on spontaneous myometrial contraction in pregnant women (between 38th and 42nd week). The IC 50 values are calculated by linear regression from cumulative concentration–response curves. HC is the highest concentration tested producing the % of relaxation indicated. Drugs
*
IC50–log (range)
% Relaxation (HC)
PPIs
PANTOPRAZOLE OMEPRAZOLE ESOMEPRAZOLE LANSOPRAZOLE RABEPRAZOLE
4.09 ( 3.24 ( 3.28 ( 3.40 ( 3.51 (
4.19/ 3.37/ 3.43/ 3.56/ 3.66/
3.98) 3.09)* 3.13)* 3.25)* 3.37)*
94 ± 5.6 45 ± 4.8 48 ± 3.5 45 ± 6.8 46 ± 3.8
(1 mM) (1 mM)* (1 mM)* (1 mM)* (1 mM)*
TOCOLYTICS
ISOXSUPRINE ATOSIBAN NIFEDIPINE MgSO4
4.05 ( 4.26 ( 6.15 ( 2.11 (
4.16/ 4.35/ 6.37/ 2.28/
3.94) 4.17) 5.92)* 1.93)*
86 ± 7.5 (0.4 mM) 93 ± 6.8 (0.15 mM) 92 ± 7.5 (10 lM) 90 ± 4.9 (2.5 mM)
p < 0.05, versus pantoprazole.
concentration used, that decreased the number of spikes in the trace (Fig. 2). The concentration–response curves relative to the % spontaneous contractility induced by pantoprazole, compared to the effect of other tocolytics (nifedipine, atosiban, isoxsuprine, MgSO4), show that the relaxing effect induced by nifedipine is reached at the lowest molar concentrations with respect to those of the other tocolytics (Fig. 3). Concentrations with the same order of magnitude are necessary for the tocolytic action induced by isoxsuprine and atosiban. Follows the pantoprazole, that, compared to the previous tocolytics, is from two to five times less potent, and finally MgSO4 that to induce a myometrium relaxation requires 8 times higher molar concentrations compared to pantoprazole. The IC50 values calculated for each agent confirm these data (Table 1) and indicate that their relative potency is the following: nifedipine o atosiban > isoxsuprine > pantoprazole o MgSO4. In a second phase of this study, by testing the pantoprazole action on myometrium sample pre-contracted by oxytocin (1 lM), PGE2 (1 lM), KCl (80 mM) and Ca++ (8 mM), it has been shown that pantoprazole at 250, 500 and 1000 lM concentration inhibits, significantly and in concentration–response manner, the contractions induced by each of the agents used (Fig. 4). The concentration of pantoprazole necessary to reduce the contractile activity of the four agents does not significantly differ, being approximately 500 lM (Fig. 4 and Table 2). Thus, this concentration of pantoprazole was chosen in the subsequent experiments of pre-treatment with antagonists. The pre-treatment with indomethacin (3 lM), L-NAME (30 lM), apamin (100 nM) and iberiotoxin (100 nM) does not modify the pantoprazole (500 lM) muscle-relaxant effect on contractions induced by KCl, oxytocin, PGE2, and Ca++, as reported in Table 2.
Fig. 2. Representative recording showing the effect of pantoprazole (500 lM) on spontaneous contractions of the myometrium of pregnant women (38–42nd week).
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Fig. 3. The cumulative concentration–response curves of known tocolytic drugs on the spontaneous contractile activity of myometrium from pregnant women (38– 42nd week) in comparison with pantoprazole. Each point represents the mean ± SE of 3 independent experiments in triplicate; ⁄ and ⁄⁄ indicate p < 0.05 and p < 0.01, respectively.
4. Discussion In the present study we demonstrate that PPIs reduce in a concentration manner the spontaneous contraction of the myometrial smooth muscle of women between 38th and 42nd week of
pregnancy, confirming that such class of drugs may play a role in the basal control of the uterine muscle tone in vitro. Pantoprazole demonstrated to be the most potent and most effective relaxant agent among the PPIs analyzed (omeprazole, esomeprazole, rabeprazole and lansoprazole). The potency order of pantoprazole muscle relaxing effect, in comparison with the other known tocolytics, is the following: significantly lower than nifedipine, approximately of the same order of magnitude as oxytocin antagonist, atosiban, and b2-agonist, isoxsuprine, but significantly higher compared to MgSO4. Pantoprazole can also reduce the contractions induced by Ca++, by the depolarizing agent KCl and by the two physiological agents of uterine contractions, oxytocin and PGE2. Furthermore, as for the possible interaction with other mechanisms underlying the relaxant effect of the PPI, our data show that the pantoprazole relaxant effect is not affected by indomethacin, the COX inhibitor, by L-NAME, the NO synthesis inhibitor, by apamin and iberiotoxin, the Ca++-dependent K+ channel blockers. Therefore, neither NO, nor PGs, or the activation of Ca++-dependent K+ currents mediate the effect of this PPI-induced decrease of myometrial tone. It is well known that PPIs inhibit the H+/K+ ATPase by a covalent modification of the enzyme (Shamburek and Schubert, 1993). H+/K+ATPase isoforms are present in organs other than the stomach including the vascular smooth muscle cells. Omeprazole, the
Fig. 4. Diagrams of miolytic effect of different concentrations of pantoprazole on contractions induced by oxytocin 1 lM (panel A), PGE2 1 lM (panel B), KCl 80 mM (panel C) and Ca++ 8 mM (panel D) in the myometrium of pregnant women (38–42nd week). Each point represents the mean ± SE of 3 independent experiments in triplicate; ⁄⁄ indicate p < 0.01. Representative recordings of effect of different pantoprazole concentrations were reported.
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Table 2 Effect of pantoprazole (500 lM) on the myometrium contraction induced by KCl, oxytocin, PGE2 and Ca++ in uterine samples of women during pregnancy (between 38th and 42nd week), in the absence and presence of different inhibitors (L-NAME, indomethacin, iberiotoxin, apamin). Drugs
% contraction induced by: KCl (80 mM)
Vehicle PANTOPRAZOLE (500 lM)
*
+Saline +L-NAME (30 lM) +INDOMETHACIN (3 lM) +IBERIOTOXIN (100 nM) +APAMIN (100 nM)
100 ± 10 38 ± 10 42 ± 12 36 ± 6 34 ± 9 40 ± 11
Oxytocin (1 lM) *
100 ± 15 32 ± 8 36 ± 8 29 ± 8 31 ± 4 38 ± 8
PGE2 (1 lM) 100 ± 12 26 ± 12 32 ± 12 25 ± 9 34 ± 10 29 ± 12
*
Ca++ (8 mM) 100 ± 9* 26 ± 9 27 ± 6 20 ± 10 19 ± 8 29 ± 10
* Indicates p < 0.01 by comparing the maximum effect (100%) induced in controls (vehicle) by the contractile agents with the one induced by the other pharmacological treatments.
H+/K+ATPase inhibitor, relaxes the spontaneous muscular tone of the experimental animal’s trachea (Rhoden et al., 1996). Furthermore, a benzimidazolic derivative (NC-1300) reduces the contractions induced by the acetylcholine in the guinea pig ileum (Okabe et al., 1996), suggesting that such derivatives can reduce the smooth muscle tone. In addition, a single work (Yildirim et al., 2001) reports that omeprazole inhibits the uterine contraction, and that the PPI relaxing effect is not mediated nor by the products resulting from the degradation in the COX nor by NO, but consequent to Ca++ channels blockers. Therefore the results of our study, on one hand confirm the data reported by Yildirim (Yildirim et al., 2001), but also extend the knowledge about such issue offering new elements for the discussion. KCl is a depolarizing agent that, by releasing acetylcholine, induces smooth muscle contraction by means of a tension-dependent intercellular Ca++ flow (Karaki, 1987). The periodic contractions of human myometrium are evoked by the release of Ca++ from intracellular deposits and by the flow through tension dependant L-type Ca++ channels (Sanborn, 2000; Wray et al., 2003). The facts that pantoprazole is capable of reducing uterine contractions induced by KCl is suggestive of an inhibiting interaction with cholinergic neuronal mechanisms, confirmed by the consequent muscle-relaxing effects in other above reported tissues (Okabe et al., 1996), and it also responsible for the intercellular Ca++ reduction. Pantoprazole relaxes the myometrium contracted by oxytocin. The contractions induced by oxytocin are due to a chemical– mechanical combination consisting in the activation of receptors associated to the phospholipase C, inducing the production of IP3 and the release of Ca++ from intracellular deposits (Giraldi et al., 1990). Furthermore, oxytocin increases the contracting system sensitivity to Ca++ signals through the rho-kinase (Bradley et al., 1998), essential for the efficacy of the combination between an increase in Ca++ and the tension developed. Once again an interference with Ca++ intracellular levels emerges among the possible targets of the PPIs muscle-relaxant action on oxytocin-induced contractions. Pantoprazole significantly reduces uterine contractions induced by PGE2. PGE2 are known as potent oxytocic agents and their action is associated to the activation of intracellular sites releasing Ca++ (Egarter and Husslein, 1992). In the myometrium of ‘‘preterm’’ and ‘‘at term’’ women in labor the COX is expressed. COX inhibitors reduce the PG production in the myometrium and are considered tocolytics (Tao et al., 2005; Choi et al., 2007). In any case it was assumed that non steroid anti-inflammatory drugs manifest their muscle relaxing effect by means of different mechanisms resulting from PG synthesis inhibition, for example by means of intercellular Ca++ reduction. On this subject, our results have shown that indomethacin, at concentrations capable of inhibiting the PG synthesis in the myometrium, does not modify the pantoprazole tocolytic action. Such datum if, on one hand, demonstrates that prostaglandinergic
pathways, and therefore the COX inhibition, do not seem to be involved in the pantoprazole tocolytic action, on the other hand, indirectly, confirms the existence of another main mediator of the contracting action by PGE2 which represents the target of the pantoprazole muscle-relaxing action on contractions induced by the PGE2, that we assume could be the intracellular Ca++. Also NO pathways are capable of relaxing animal and human myometrium (Bradley et al., 1998) but, as recently demonstrated (Buxton et al., 2001), not through the cGMP activation, responsible for mediating most of the actions of this new neurotransmission system, but rather by means of the direct or indirect activation of K+ channels through Ca++. Since the relaxation induced by NO donors can be prevented by some toxins, specific inhibitors of these high conductance channels for K+, it is highly probable that one more of these channels, as expressed in the human preterm myometrium, can be the target for NO and other tocolytics. The mechanism regulating the muscle-relaxant action would consist in an increase in intracellular Ca++, responsible for the K+ channel activation associated to Ca++, whose opening would induce the activation of tension-dependent channels permeable to K+. It could provoke cell hyperpolarization and consequently relaxation. Actually such conceivable mechanism has not been demonstrated to take place in myometrium. In any case, about this issue, the results, we obtained, demonstrate that in the muscle relaxing action induced by pantoprazole, neither NO nor K+ channels activated by Ca++ play the role of mediators. The pre-treatment of myometrium preparation, both with NO synthesis inhibitor or K+ channel blockers, apamin and iberiotoxin, does not modify the smooth muscle-relaxing effect of this PPI on oxytocin- induced contractions. All these considerations suggest that PPIs induce a smooth muscle relaxing effect on the gravid uterus probably using a target common to other systems and to different endogenous mechanisms capable of controlling the contracting and/or relaxing capacity of the uterine smooth muscular fibers. Most of the tocolytic agents regulate the intracellular Ca++ concentration although the function of such channels during delivery is still unknown. The beginning of delivery is associated to many myometrial modification including alterations in electrophysiological mechanisms (gap junctions, membrane ion channels) (Miller et al., 1989; Garfield et al., 1992) and biochemical processes (actin/myosin coupling, receptors) controlling myometrium contractility and relaxation (Egarter and Husslein, 1992; Hertelendy and Zakar, 2004). For example, it has been recently observed that in the myometrial cells of gravid rats during the last period of gestation the expression of rapid Na+ channels increases more than the expression of slow Ca++ channels (Ohya and Sperelakis, 1989; Sperelakis et al., 1992). Also K+ channels play a role in the regulation of uterine contractility, and modifications in the expression and in the function of such channels can be responsible for myometrial modifications during the different phases of the pregnancy (Lundgren et al., 1997; Knock et al., 1999; Brainard et al., 2005).
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The fact that pantoprazole has a relaxing effect on uterine contractions induced by different contracting agents but all using Ca++ as final mediator of their action, let us assume, by deduction, that PPIs may interfere with the intracellular concentration of this ion by reducing it even if we are not able to define the different phases leading to the intracellular Ca++ reduction. Nevertheless, we cannot exclude that other signal mechanisms regulating smooth muscle contraction may intervene in the pantoprazole-induced inhibition. Furthermore, the data of this study do not permit to define whether the PPIs action is specific or non specific. It is important to remark that the PPIs receptor site of action, the H+/K+ ATPase, has a high level of structural homology with Na+/K+ ATPases in certain tissue such as colon, presenting a similar pharmacologic profile as both are sensitive to ouabain. Such ATPases can transport Na+ outside the cell exchanging it with K+ due to its sensitivity to cations. It has been recently demonstrated (Veklich et al., 2007) that omeprazole and lansoprazole (10–100 lM) inhibit also the activity of such pump at plasma membrane level in myometrial smooth muscle cells with a consequent reduction in Na+/K+ exchanges, an increase in intracellular K+ and a reduction in cell excitability. However, the lack of data clearly indicating the function of both proton pumps in human myometrium does not allow us to accurately define the mechanism regulating the muscle relaxant action in PPIs. 5. Conclusions To conclude, this study clearly confirms the relaxant effect of PPIs in human myometrium in vitro. All the PPIs, tested at sufficient concentration, are capable of relaxing the myometrial tissue samples even if their potency compared to other well known tocolytics is almost always lower. Notwithstanding, the pharmacological potency relative to the muscle relaxant action tested in vitro does not correspond to a clinic tocolytic efficacy, but it merely indicates that a certain drug is more effective at a lower dose. The extrapolation of the concentration in a bath for isolated tissues at plasmatic level in patients is full of uncertainties. First of all, it has been demonstrated that the plasmatic levels of some of this tocolytic agents widely vary due to differences in their pharmacokinetics and they are not always correlated with their muscle relaxant action. Secondly, similar concentrations in vitro and in the patients’ plasma in vivo do not correspond to similar concentrations in the tissues. Usually, clinical studies on different tocolytics demonstrated that the concentrations in vitro were higher than plasmatic concentrations in vivo. Such discrepancy could be the cause of the ineffectiveness of some tocolytics in preventing preterm delivery. Furthermore, in the clinical practice, toxicity is a limit for the plasmatic concentrations to be reached. Most of the time-concentrations producing a muscle relaxing effect can be associated to severe side effects. As the PPIs almost do not induce side effects means that such drugs can offer new therapeutic strategies even if, undoubtedly, further investigations to understand the mechanism regulating their tocolytic action and clinical studies in vivo are necessary before adding PPIs to the list of drugs available for the treatment of preterm delivery. References Bradley, K.K., Buxton, I.L., Barber, J.E., McGaw, T., Bradley, M.E., 1998. Nitric oxide relaxes human myometrium by a cGMP-independent mechanism. Am. J. Physiol. 275, C1668–C1673. Brainard, A.M., Miller, A.J., Martens, J.R., England, S.K., 2005. Maxi-K channels localize to caveolae in human myometrium: a role for an actin-channelcaveolin complex in the regulation of myometrial smooth muscle K+ current. Am. J. Physiol. Cell Physiol. 289, C49–C57. Buxton, I.L., Kaiser, R.A., Malmquist, N.A., Tichenor, S., 2001. NO-induced relaxation of labouring and non-labouring human myometrium is not mediated by cyclic GMP. Br. J. Pharmacol. 134, 206–214.
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