Molecular Human Reproduction, Vol. 9, No. 3, 159-164,
March 2003
© 2003 European Society of Human Reproduction and Embryology
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Effects of L-type Ca2+-channel blockade, K+ATP-channel opening and nitric oxide on human uterine contractility in relation to gestational age and labour
Submitted on October 11, 2002; accepted on December 13, 2002
Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, TX 77555, USA
1 To whom correspondence should be addressed at: Division of Reproductive Sciences, Department of Obstetrics and Gynecology, The University of Texas Medical Branch, 301 University Blvd., Rt. J-62, Galveston, TX 77555-1062, USA. e-mail: rgarfiel{at}utmb.edu
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ABSTRACT |
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Relative uterine inactivity during pregnancy changes to vigorous rhythmic contractility during labour. We hypothesized that mechanisms involved in the regulation of uterine quiescence and contractility differ between term and preterm myometrium and in labour and non-labour states. Myometrial strips, prepared from biopsies taken at Caesarean section from labouring and non-labouring women preterm and at term, were mounted in organ chambers for isometric tension recording. Oxytocin (10–9 mol/l) was added to maintain stable contractions, and effects of various inhibitors of uterine contractility were studied. The inhibitory effects of L-type Ca2+-channel blocker nifedipine and ATP-sensitive K+-channel opener pinacidil were greater in myometrium from the non-labour versus the labour group, both preterm and at term. In addition, pinacidil’s effect was greater at term compared with preterm in the non-labour group. Mg2+ and the nitric oxide donor sodium nitroprusside significantly inhibited contractility in all groups without significant differences with regard to labour or gestational age. Decreased inhibition of human uterine contractility by L-type Ca2+-channel blockers and K+ATP-channel openers in preterm and term labour may reflect changes in expression and activity of these channels. Effects of nitric oxide and Mg2+ are not affected by gestational age or labour.
Key words: channels/human/labour/pregnancy/uterus
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Introduction |
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The incidence of preterm labour remains unchanged despite significant advances in the understanding of uterine physiology (Challis, 2000). Several agents are used clinically as tocolytics including magnesium sulphate, cyclooxygenase inhibitors (e.g. indomethacin), nitric oxide donors (e.g. nitroglycerine) and Ca2+-channel blockers (e.g. nifedipine) (Childress and Katz, 1994; Lees et al., 1994; El-Sayed et al., 1999). Uterine contractile activity is determined by the increase in intracellular free Ca2+ concentration in the myometrial cells. Ca2+-Channel blockers inhibit contractions by decreasing Ca2+ influx (Barany, 1996). Mg2+ also inhibits Ca2+ entry into the myometrial cell through Ca2+-channels, though it may have other intracellular effects (Iseri and French, 1984; Altura et al., 1986). K+-channel activation has an inhibitory effect on uterine contractile activity through hyperpolarization of myometrial cells, and K+-channel openers may have a potential use as tocolytics (Morrison et al., 1993; Okawa et al., 1999). Alteration in expression and function of Ca2+- and K+-channels may play a role in the onset of labour (Mironneau, 1994; Tezuka et al., 1995; Khan et al., 2001).
Data from our laboratory, as well as others’, support the presence of a functional L-arginine–nitric oxide (NO) pathway in myometrium (Izumi et al., 1993; Buhimschi et al., 1995). NO, a potent smooth muscle relaxant, is synthesized from L-arginine by nitric oxide synthase (NOS) (Moncada et al., 1991; Moncada and Higgs, 1993). The down-regulation of the NO-soluble guanylate cyclase (sGC)–cyclic guanosine monphosphate (cGMP) system in the uterus at term, and the decreased sensitivity to NO, precede the appearance of forceful uterine contractions in preterm and term labour (Yallampalli et al., 1994; Buhimschi et al., 1995, 1996). The inhibitory effect of NO, as well as other ion channels, may be important in the maintenance of uterine quiescence during pregnancy; in which case, the inhibitory effects of these inhibitory mechanisms may be diminished as pregnancy progresses or labour ensues. The aim of this study was to investigate the effect of gestational age and labour on the effectiveness of various inhibitors on uterine contractile activity in the human uterus.
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Materials and methods |
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In-vitro contractile activity of myometrial strips from pregnant women with or without labour at preterm and term was studied. The protocol was approved by the Institutional Review Board for Human Research of the University of Texas Medical Branch. All patients gave informed consent.
Human tissue
Myometrial tissue was obtained from pregnant women at preterm and term pregnancy undergoing Caesarean section under epidural anaesthesia during labour or prior to the onset of labour (Table I). Biopsies were taken from the upper edge of the transverse incision in the lower uterine segment, placed in Hanks’ balanced salt solution (HBSS; Gibco BRL Products, Rockville, MD, USA), and transported to the laboratory where they were transferred to Krebs–Henseleit solution and prepared into 10x1.5x1.5 mm strips. Tissues with irregular appearance evident during dissection or poor contractile activity obtained from women undergoing repeat Caesarean sections were discarded with the assumption that they represented scar tissue.
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Drugs and solutions
The agents used in the experiments were L-arginine, nifedipine, oxytocin, sodium nitroprusside (Sigma, St Louis, MO, USA) and pinacidil (RBI, Natick, MA, USA). Stock solutions of L-arginine and sodium nitroprusside (10–1 mol/l) were prepared in de-ionized water, aliquoted and kept at –20°C until use. Oxytocin was dissolved in distillated water (10–3 mol/l) and sonicated for 5–15 min to aid solubilization. Nifedipine and pinacidil were dissolved in dimethylsulphoxide (DMSO) (10–1 mol/l). The composition of Krebs–Henseleit solution (in mmol/l) was NaCl 119, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, CaCl2 2.5, glucose 11.5 and EDTA 0.026.
In-vitro experiments
The myometrial strips were mounted vertically in 10 ml organ chambers containing Krebs–Henseleit solution and prepared for isometric tension recording using stainless steel hooks and surgical thread. One end of the strips was attached to a fixed support at the bottom of the chamber, while the other end was connected to an isometric force transducer. The temperature in the organ bath was thermostatically maintained at 37°C and the solution was continuously bubbled with 5% CO2 in air (pH 
7.4). Strips were equilibrated at the passive tension of 1 g in Krebs’ solution. Isometric tension was measured with Harvard isometric force transducers (Harvard Apparatus, South Natik, MA, USA) connected to an on-line computer. The data were acquired, stored and analysed using Windaq data acquisition and playback software (Dataq Instruments, Inc., Akron, OH, USA).
The strips were equilibrated for 1–2 h until the contractions stabilized. The bath solution was changed every 30 min during the equilibration period. The presence of infrequent spontaneous contractions in human tissues, especially in the non-labour state, limits the ability to study the effects of inhibitory agents. Therefore, oxytocin (10–9 mol/l) was added to the organ chambers for all the preparations to maintain a more uniform pattern of uterine contractile activity. Although oxytocin may affect some myometrial regulatory pathways differently, appropriate temporal controls were used for each study group. Oxytocin and the solvent for the inhibitory agents (e.g. DMSO or Krebs’ solution) were added to these control preparations at the same time as oxytocin or the inhibitory agents were added to study preparations. The contractile activity in the controls was measured in parallel to the study preparations (Figure 1). The effects of cumulative concentrations of pinacidil (ATP-sensitive K+-channel opener, 10–8 to 10–4 mol/l), nifedipine (L-type Ca2+-channel blocker, 10–8 to 10–5 mol/l), Mg2+ (2 to 16 mmol/l), L-arginine (NO synthase substrate, 10–6 to 10–3 mol/l), and sodium nitroprusside (SNP, NO donor, 10–8 to 10–4 mol/l) were then studied.
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Data analysis
The uterine contractility was estimated by calculating the integral activity (area under the uterine tracing) of uterine strips for a 20 min period. The basal activity was taken as the integral activity over 20 min starting 30 min after the addition of oxytocin and prior to the addition of the studied agents. The effect of cumulative concentrations of the inhibitors was determined by calculating the integral activity expressed as percentage change from the basal integral activity over 20 min after each cumulative dose of the agent. The effect of the various inhibitory agents at each concentration was calculated as a percentage decrease from the oxytocin-induced contractile activity. The temporal controls run in parallel with each concentration–response experiment were used to normalize for the effects of the solvents or decay in contractility with time. The decrease in the contractile activity in the temporal controls was subtracted from the concentration–responses to the inhibitory agents.
The decrease in the contractility in these controls was subtracted from the respective concentration–response data. The area under the concentration– response curve (AUC for Figures 1–5) and the negative logarithm of the concentration producing 50% inhibition (–log IC50; a measure of sensitivity) and the maximal effect were calculated for each agent. The AUC and the –log IC50 were performed using GraphPad Prism version 3.00 for Windows, (GraphPad Software, San Diego, CA, USA). One-way analysis of variance followed by Student–Newman–Keuls’ method and Mann–Whitney U-test were used, as appropriate, for statistical analysis, and P < 0.05 was considered statistically significant. The data are expressed as the mean ± SEM. The n value represents the number of samples (each from a different patient) used in each experiment.
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Results |
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The frequency of contractions in the human myometrium in the four study groups after stimulation with oxytocin was not significantly different [frequency (contractions/h): term non-labour 19.0 ± 2.4, term labour 22.1 ± 3.0, preterm non-labour 14 ± 1.9, preterm labour 21.6 ± 3.3].
The inhibitory effect of pinacidil on human myometrial contractility was greater in the tissues from term non-labour (NLterm) pregnant women as compared with term labour (Lterm) as evidenced by comparison of AUC and maximal effect. Also the –log IC50 in the Lterm group shifted to the right compared with the preterm non-labour (NLpreterm) group (Figure 2, Table II). In tissues obtained from preterm women, only the maximal effect was significantly lower in the preterm labour (Lpreterm) group compared with the preterm non-labour (NLpreterm) group (Figure 2, Table II). In the NLterm group, the maximal inhibition induced by pinacidil was also significantly higher compared with Lterm, NLpreterm and Lpreterm groups (Figure 2, Table II).
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The inhibitory effect of nifedipine on the myometrial strips was less pronounced in Lterm group compared with NLterm group in terms of AUC (Figure 3, Table III). The maximal effect in the Lpreterm group was significantly lower compared with all the groups. The effects of nifedipine in strips from NLpreterm and NLterm groups did not differ significantly.
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Mg2+ significantly inhibited the contractile activity of myometrial strips from NLpreterm, Lpreterm, NLterm and Lterm groups in a concentration-dependent manner without any significant differences in AUC or maximal effect between the groups (maximal effect: 93.8 ± 8.1%, 87.3 ± 8.0%, 80.3 ± 7.2% and 99.2 ± 13.0% respectively; Figure 4). The inhibition induced by L-arginine in the four groups was negligible; the maximal inhibition was 19.4 ± 5.8%, 27.9 ± 17.8%, 12.3 ± 4.7% and 32.1 ± 7.4% in NLpreterm, Lpreterm, NLterm and Lterm groups respectively (Figure 5). SNP significantly inhibited contractility in myometrial strips from NLpreterm, Lpreterm, NLterm and Lterm groups without significant differences between them in terms of AUC, maximal effect or –log IC50 (maximal effect: 59.0 ± 6.5%, 51.5 ± 17.7%, 82.0 ± 10.1% and 55.5 ± 4.4% respectively; Figure 6).
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Discussion |
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The onset of labour, both term and preterm, is preceded by a preparatory phase during which the uterus changes from a relatively inactive to a vigorously active organ in terms of contractility (Garfield et al., 1998). The present study demonstrates that the onset of either preterm or term labour is associated with a decreased ability of pinacidil, the ATP-sensitive K+-channel opener, and nifedipine, the L-type Ca2+-channel blocker, to inhibit uterine contractility of isolated human myometrium in vitro. In contrast, the effectiveness of Mg2+ and the NO donor SNP remains unaltered.
Oxytocin was used to stimulate the contractility of the myometrial preparations before studying the effects of the various inhibitors. Varying effects of oxytocin at different stages of pregnancy and in the absence or presence of labour may have affected our results. However, differences were noted in the effects of pinacidil and nifedipine, but not those of Mg2+ or NO. Therefore, it is unlikely that the differences noted were due to the use of oxytocin.
Opening of K+-channels in the myometrial cell is accompanied by cell membrane hyperpolarization (Barany, 1996). This hyperpolarization impedes Ca2+ entry into the cell through voltage-gated L-type Ca2+-channels, thereby exerting an inhibitory influence on uterine activity (Barany, 1996; Okawa et al., 1999). Decreased uterine contractility in human myometrium by the ATP-sensitive K+-channel opener pinacidil was greater at term compared with preterm gestation. This may indicate an increased inhibitory influence of the ATP-sensitive K+-channels on the myometrium with advancing gestation that may be important for maintenance of uterine quiescence. The effect of pinacidil was decreased during preterm and term labour compared with the non-labour state. A decreased inhibitory effect of the ATP-sensitive K+-channels may be necessary for forceful uterine contractions during preterm and term labour, and may result from increase in myometrial depolarization, as well as increased Ca2+-channel density and Ca2+ entry before labour (Tezuka et al., 1995). Thus a decrease in the inhibitory effect of ATP-sensitive K+-channels on uterine contractility may be a prerequisite for the onset of labour.
An increase in intracellular free Ca2+ is the trigger for myometrial contractions (Anwer and Sanborn, 1989). Ca2+-Channels are important for Ca2+ entry from the extracellular space as well as Ca2+ release from the intracellular stores (Barany, 1996). Nifedipine, an L-type Ca2+-channel blocker, has been shown to inhibit uterine contractions in several studies in animals and humans (Childress and Katz, 1994). We found that the inhibitory effect of nifedipine was decreased in preterm or term labour compared with the corresponding non-labour state. In addition, all the groups except term labour were sensitive to very low concentrations of nifedipine. These data indicate that nifedipine is an inhibitor of myometrial contractile activity in the non-labour state as well as in preterm labour; however, term labour has increased resistance to its effects. This may indicate changes in the L-type Ca2+-channel function during pregnancy and in labour. The number and sensitivity of the Ca2+ channels increase in labour, making them more resistant to their physiological or pharmacological inhibitors, hence allowing greater Ca2+ influx and more forceful contractions during labour (Tezuka et al., 1995). In contrast to nifedipine, the effectiveness of Mg2+ in inhibiting uterine contractions remained unchanged with advancing gestation or with onset of preterm or term labour. Increasing concentrations of Mg2+ as used in the current study protocol in vitro do not alter the extracellular Ca2+ levels. Although action of Mg2+ may be mediated through Ca2+-channels, Mg2+ is a less specific Ca2+-channel antagonist and may have additional intracellular effects (Iseri and French, 1984; Altura et al., 1986).
A number of studies have shown that the L-arginine–NO–cGMP pathway is present in the rat, guinea-pig and human uterus, and that its activation causes a substantial inhibition of uterine contractility (Izumi et al., 1993; Buhimschi et al., 1995; Facchinetti et al., 1996). The inducible NOS isoform (iNOS), which is present in a variety of cells in the myometrium, including the uterine smooth muscle, macrophages and trophoblast cells, is the source of endogenous NO (Wu, 1995; Gangula et al., 1997). In addition, NO is produced by the endothelial NOS isoform (eNOS) that is expressed in the vascular endothelium (Wu, 1995). However, the role of NO in controlling uterine activity still remains unclear (Hennan and Diamond, 1998). This pathway may contribute to maintenance of relative uterine quiescence during pregnancy and its down-regulation at term may be important for the onset of labour (Izumi et al., 1993; Buhimschi et al., 1995). In addition, the sensitivity of the rat myometrium to NO donors has been demonstrated to decrease at term (Buhimschi et al., 1995, 1996). In the present study, the NOS substrate L-arginine given in millimolar concentrations did not have a significant effect on uterine activity in any of the groups studied. This may indicate that adequate levels of L-arginine are maintained in the myometrium and that it may not be a rate-limiting factor for the synthesis of NO. NO donors have been used in the management of preterm labour or other conditions that require myometrial relaxation (Lees et al., 1994; El-Sayed et al., 1999). We have previously shown that NO inhibits spontaneous contractile activity in isolated strips from human myometrium at term (Longo et al., 1999). Ekerhovd et al. (1999) showed that NO inhibits myometrial contractile activity, and that the responsiveness to NO was similar in non-labouring and labouring women.
In this study, we confirmed that the sensitivity of human myometrium to NO does not change in relation to gestational age or with the onset of preterm or term labour. Therefore, in humans, a decrease in the sensitivity of the myometrium to NO does not appear to contribute significantly to the onset of preterm or term labour. However, it remains to be investigated whether a change in the NO synthesis occurs at the time of labour in humans.
This study also showed that the inhibitory activity of Mg2+ and the NO donor SNP on uterine strips at preterm or term gestation does not decrease with the onset of labour. These findings are consistent with the clinical effectiveness of these agents for management of preterm labour as well as for obtaining uterine relaxation in various obstetric emergencies such as retained placenta, difficult fetal extraction during Caesarean delivery and uterine inversion (El-Sayed et al., 1999). The study also confirmed that regulation of the expression and function of ATP-sensitive K+-channels and L-type Ca2+-channels may play a significant role in preparation of the uterus for preterm or term labour.
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