Inhibition of nitric oxide synthesis causes preterm delivery in the mouse

Gian Mario Tiboni1 and Franca Giampietro

Sezione di Ostetricia e Ginecologia, Dipartimento di Medicina e Scienze dell'Invecchiamento, Facoltà di Medicina e Chirurgia, Università `G. d'Annunzio', Chieti, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of the present study was to investigate whether an inhibitor of nitric oxide (NO) synthesis provokes preterm delivery in a mouse model. ICR (CD-1) mice were injected s.c. with NG-nitro-L-arginine methyl esther (L-NAME) at 0, 40, 70 or 100 mg/kg on gestation day (GD) 15.5 and 16. Delivery was considered preterm if it occurred before GD 18. In a satellite study, the potential ability of the NO donor sodium nitroprusside (SNP) to prevent L-NAME-induced preterm delivery was tested. Five hours before the initiation of treatment regimen with L-NAME at 70 mg/kg, mice were implanted s.c. with micro-osmotic pumps infusing SNP at 0 or 10 µg/kg/min continuously for 3 days. Administration of L-NAME evoked preterm delivery. This response was noted in 64 and 60% of animals treated with 70 and 100 mg/kg L-NAME respectively (P < 0.05 versus control value). Infusion with SNP provided complete and significant (P < 0.05 versus positive control value) protection from L-NAME-initiated preterm delivery. This is the first report to reveal that an inhibitor of NO synthesis initiates preterm delivery in a mouse model.

Key words: L-NAME/mouse /nitric oxide/preterm delivery


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prevention of preterm delivery is a major undertaking in human health. Deliveries ending before term account for about 10% of all deliveries but for 75% of neonatal mortality and 50% of long-term neurological sequelae (Creasy, 1993Go). It is generally agreed that the unavailability of pharmacological interventions effective in preventing preterm labour and resultant preterm delivery probably reflects the poor understanding of molecular mechanisms behind the initiation and maintenance of labour.

The free radical and potent smooth muscle relaxant nitric oxide (NO) is synthesized by a family of enzymes known as NO synthases (NOS), which catalyse the conversion of L-arginine to NO plus citrulline (Moncada et al., 1995Go). As is well established, NOS activity can be competitively inhibited by a variety of L-arginine analogues (Moncada et al., 1995Go). NO has been recently postulated as having a role in the complex molecular interplay which regulates myometrial function during gestation (Norman, 1996Go; Sladek et al, 1997Go; Yallampalli et al., 1998Go), and several animal and human studies have been conducted on this subject during the last few years.

Evidence exists that generation of NO occurs in uterine tissues of several species, including the rat (Conrad et al., 1993Go; Izumi et al., 1993Go; Natuzzi et al., 1993Go; Yallampalli et al., 1993Go, 1994Go), guinea-pig (Weiner et al., 1994Go), rabbit (Sladeck et al., 1993), sheep (Figueroa and Massmann, 1995Go) and mouse (Huang et al., 1995Go; Moorhead et al., 1995Go). In the rabbit (Sladeck et al., 1993) and rat (Yallampalli et al., 1993Go), NO synthesis showed a phase-dependency, being up-regulated during gestation and down-regulated during labour. Notably, the contractility of pregnant rat uterine strips was inhibited by NO and its precursor L-arginine (Yallampalli et al., 1993Go; Izumi et al., 1993Go). The responsiveness of the rat uterus to the relaxant effect of NO was found to be decreased during labour (Yallampalli et al., 1993Go; Buhimschi et al., 1995bGo).

Studies on the human pregnant uterus have documented myometrial NOS activity (Telfer et al., 1995Go; Ramsey et al., 1996; Thomson et al., 1997Go; Bansal et al., 1997Go; Ekerhovd et al., 1999Go). The ability of exogenous NO to evoke myometrial relaxation has also been determined (DeSimone et al., 1990Go; Greenspoon and Kovacic, 1991Go; Buhimschi et al., 1995aGo; Lee and Chang, 1995Go). On the other hand, contrary to what has been observed in animal models, myometrial tissue strips obtained from pregnant women were not relaxed by L-arginine (Jones and Poston, 1997Go; Ekerhovd et al., 1999Go). A decline in human myometrial NOS expression in association with labour and delivery has been reported (Bansal et al., 1997Go).

Despite the large number of functional and molecular studies, the exact role of the NO system in the control of myometrial function during pregnancy has not yet been clearly defined. We found intriguing the lack of substantial evidence that inhibition of NO synthesis can precipitate parturition (Norman, 1996Go; Sladek et al., 1997Go; Yallampalli et al., 1998Go). This study therefore aimed to address this investigative shortcoming by determining the potential capacity of a competitive inhibitor of NOS to trigger delivery in a mouse model, which has not to our knowledge been done before.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and breeding procedure
Sexually mature ICR (CD-1) outbred mice (Harlan Italy, Udine, Italy) were used in the study. Animals were kept under controlled conditions (room temperature of 22 ± 1°C; 55 ± 5% of relative humidity) on a 12 h light/dark cycle, with rodent laboratory chow (Harlan Teklad®; Harlan Italy) and municipal water provided ad libitum. After an acclimatization period of a week, females (three to four per cage) were co-habited overnight with males of the same stock and source, and examined for the presence of a copulatory plug at the end of the dark cycle (0800 h). The day on which the plug was observed was designated as gestation day (GD) 0.

Study 1
The L-arginine analogue NG-nitro-L-arginine methyl esther (L-NAME), a competitive NOS inhibitor, was purchased from Research Biochemicals International (Natick, MA, USA). Dosing solutions were prepared by dissolving the L-NAME in sterile and apirogenic saline solution. On GD 15.5 (2000 h of gestation day 15) and 12 h later, on GD 16, animals were dosed with 0 (vehicle), 40, 70 or 100 mg L-NAME/kg body weight. The test article was injected s.c., in the interscapular region, using a 26 gauge needle. The volume administered was 10 ml/kg body weight. By observation of animals at intervals of 2–4 h, the timing of delivery was determined. Delivery was considered preterm if occurring before GD 18. In order to establish whether or not deliveries occurring before term were giving birth to all fetuses, maternal laparotomy was performed 12 h after the occurrence of parturition was noted. All term and preterm newborns were inspected for viability.

Study 2
This study was undertaken to evaluate the potential ability of the NO donor sodium nitroprusside (SNP; Sigma Chemical Co., St. Louis, MO, USA) to prevent L-NAME-induced preterm delivery. Five hours before the initiation of the treatment regimen with L-NAME at 70 mg/kg, animals were implanted s.c. (in the lumbar region of the back) with a micro-osmotic pump (model 1035D; Alza, Palo Alto, CA, USA), delivering SNP at 10 µg/kg/min continuously for 3 days. Control animals were implanted with pumps filled with vehicle (saline solution). The implantation procedure was carried out under a light general anaesthesia induced by ether inhalation. Sterile stainless steel skin staplers were used to close implantation wounds. The same endpoints of study 1 were determined.

Statistical analysis
Fisher's exact test was used to evaluate differences between various experimental groups. The results were considered to be significant when the P value was < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study 1
As shown in Figure 1Go, all control animals (n = 13) delivered at gestational term (>=GD 18). On the other hand, treatment with L-NAME caused mice to deliver before term (Figure 1Go). This response was noted in approximately 18, 64 and 60% of pregnant mice injected with 40 (n = 11), 70 (n = 14) and 100 (n = 10) mg L-NAME/kg respectively. It must be noted, however, that only incidences recorded in groups treated with 70 and 100 mg/kg were statistically significant (P < 0.05) compared with the control value (Figure 1Go). Table IGo details the timing of delivery. As can be seen, the majority of preterm deliveries occurred between GD 16.5 and GD 17.5, and only two animals (in the group exposed at the maximal level of L-NAME) delivered at an earlier time (on GD 16–16.5). Intriguingly, preterm delivery never occurred between GD 17.5 and GD18. Maternal laparotomies revealed that delivery, irrespective of the timing of occurrence, always gave birth to the entire litter (data not shown). It was noted that the threshold dose (70 mg L-NAME/kg) for a significant elicitation of preterm delivery had no noticeable clinical effect on maternal wellbeing. On the other hand, a light and transient depression of spontaneous movement followed treatment with L-NAME at 100 mg/kg.



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Figure 1. Effect of L-NAME on gestation duration in ICR (CD-1) mice. Animals were injected s.c. with L-NAME on GD 15.5 and 16. Delivery was considered preterm if occurring before GD 18. The numbers in parentheses indicate the ratio of animals delivering preterm to total animals treated. *Significantly different compared with control group (P < 0.05).

 

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Table I. Effect of L-NAMEa on the timing of delivery in ICR (CD-1) mice
 
The complete evaluation of neonatal viability was precluded by maternal cannibalism of newborns. Despite this investigative shortcoming, the overall impression was that the vitality of pups was related to the timing of delivery. In this regard, whereas offspring delivered on GD 16 invariably appeared dead (often still surrounded by membranes and placenta), several pups delivered on GD 17 appeared alive when inspected immediately after delivery, but not later. On the other hand, when the gestation ended at term, offspring of mothers treated with L-NAME at various doses exhibited a viability that seemed comparable to that of controls (data not shown).

Study 2
As illustrated in Figure 2Go, three of eight (37.5%) animals implanted with vehicle-filled pumps delivered preterm. In all three instances, gestations ended on GD 16.5–17 (Table IIGo). By contrast, mice infused with SNP at 10 µg/kg/min for 3 days (n = 12) showed a complete and statistically significant (P < 0.05 versus positive control group) protection against the shortening effect of L-NAME at 70 mg/kg on gestation length (Figure 2Go). No clinical signs of maternotoxicity were observed in animals used in this study.



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Figure 2. Effect of sodium nitroprusside (SNP) infusion on L-NAME-initiated preterm delivery. L-NAME was injected s.c. at 70 mg/kg on GD 15.5 and 16. Delivery was considered preterm if it occurred before GD 18. Five hours before the initiation of the treatment regimen with L-NAME, animals were implanted s.c. with micro-osmotic pumps delivering SNP at 10 µg/kg/min continuously for 3 days. Control animals were implanted with micro-osmotic pumps filled with vehicle (saline solution). The numbers in parentheses indicate the ratio of animals delivering preterm to total animals treated. *Significantly different compared with control group (P < 0.05).

 

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Table II. Effect of SNPa infusion on L-NAMEb-induced preterm delivery in ICR (CD-1) mice
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Research conducted in the past decade has established the ability of the human myometrium to generate NO (Telfer et al., 1995Go; Ramsey et al., 1996; Bansal et al., 1997Go; Thomson et al., 1997Go; Ekerhovd et al., 1999Go; Norman et al., 1999Go), and to relax in response to exogenous NO (DeSimone et al., 1990Go; Greenspoon and Kovacic, 1991Go; Lee and Chang, 1995Go; Buhimschi et al., 1995aGo). These findings have provided impetus for new research, with the primary aim of developing effective tocolytic approaches. Unfortunately consensus regarding the role of NO system in the control of myometrial contractility is lacking (Jones and Poston, 1997Go).

In the search for additional information on mechanisms involved in uterine quiescence and contractility, we found in this study that treatment with L-NAME leads to preterm delivery in the mouse. This is to our knowledge the first time that a NOS inhibitor has been found per se to terminate gestation in a rodent model. The threshold dose for such a response was 70 mg L-NAME/kg given twice on GD 15.5 and GD 16, e.g. at a gestational stage when the murine conceptus is terminating organogenesis and is entering the fetal period of antenatal life. The effect of L-NAME on gestation duration did not fit a dose-response model, as shown by the fact that preterm delivery incidences following administration of 70 or 100 mg/kg were comparable. However, a possible dose-dependency is suggested by the low rate of preterm delivery observed (although not significant) in the experimental group treated with the lowest dose of L-NAME (40 mg/kg), and by the fact that a trend toward an earlier timing of delivery with the increment of L-NAME concentration was seen.

SNP is a drug that, without requiring enzymatic bioactivation, is readily converted to NO in biological systems (Sladek et al., 1997Go). This NO donor has been found, albeit with some inconsistencies, to effect relaxation of pregnant myometrium (Sladek et al., 1997Go). In the present study SNP was infused at a rate (10 µg/kg/min) that is within the human therapeutic zone (although at the higher extreme) (Gerber and Nies, 1990Go) and it was found to provide complete protection from preterm delivery. This result suggests that L-NAME evoked preterm delivery via inhibition of NO synthesis and not by alternative pharmacological or toxicological mechanisms. Biological plausibility for this hypothesis is also provided by the observation that the mouse uterus is a NO-producing organ (Huang et al., 1995Go; Moorhead et al., 1995Go), and that uterine horns of pregnant mice exhibited isometric tension increments after application of a NOS inhibitor (Boquet et al., 1998Go).

Over the last decade, a number of investigations (Yallampalli and Garfield, 1993; Diket et al., 1994Go; Molnar et al., 1994Go; Buhimschi et al., 1995bGo; Salas et al., 1995Go; Helmbrecht et al., 1996Go; Yallampalli et al., 1996Go) have been made into the gestational response to inhibitors of NO synthesis in rats. In these investigations, L-NAME and other L-arginine analogues were found to induce fetal growth restriction with (Yallampalli and Garfield, 1993; Molnar et al., 1994Go; Buhimschi et al. 1995bGo; Helmbrecht et al., 1996Go; Yallampalli et al., 1996Go) or without (Diket et al., 1994Go) other signs evocative of a pre-eclampsia-like syndrome, but not preterm delivery. At present, to our knowledge, the only existing evidence for a cause-effect relationship between inhibition of NO synthesis and preterm delivery is represented by an investigation carried out on the guinea-pig (Chwalisz et al., 1996Go). It is interesting to note in that study that administration of L-NAME, possibly due to an impairment of cervical ripening, besides shortening gestation length also caused a prolongation of the delivery process (Chwalisz et al., 1996Go). This result appears consistent with the view that NO is relevant to the process of natural cervical ripening (Buhimshi et al., 1996; Thomson et al., 1996Go; Chwalisz et al., 1997Go; Tschugguel et al., 1999Go) and has contributed to the recent proposal that for successful gestation and parturition a spatial regulation in NOS activity/expression must occur in uterine tissues. While during gestation NO generation would be up-regulated in myometrium and down-regulated in the cervix, assuring uterine relaxation and cervical competence, the opposite effects would occur during labour, leading to myometrial contraction and cervical ripening (for review see Chwalisz and Garfield, 1988, 1998). In this regard, it is possible that the relative contribution of the NO system in this dual role may significantly differ depending on the species, and that interspecies variations may partly account for the discrepant responses to NO inhibitors noted between the rat, guinea pig and mouse.

Besides the putative role played by a species-specific intrinsic sensitivity, it is also conceivable that the ability of L-NAME to terminate the mouse pregnancy is dependent on the experimental conditions selected. This is also apparent from results of a pilot study carried out in our laboratory (unpublished observations) where ICR (CD-1) mice received L-NAME in drinking water at an estimated daily dose of 0 (n = 8), 140 (n = 9) or 280 (n = 9) mg/kg/day. Exposure was continued from GD 14–18 (term gestation) when pregnancies were terminated and several fetal end-points evaluated. Under these circumstances, L-NAME was found to retard the fetal growth process, but not to elicit preterm delivery. Therefore it may be that initiation of preterm delivery by L-NAME requires a particular pattern of NO system down-regulation, and that this condition is achieved with bolus injection but not with chronic administration. In other words, from a pharmacokinetic point of view, it would seem that the maximal concentration reached by L-NAME in the systemic circulation is a more critical parameter for initiation of preterm delivery than the area under the concentration-time curve, a parameter indicative of total exposure. This idea seems worthy of further investigation.

Collectively, data obtained in this investigation are consistent with the idea that the NO system may play a role in the regulation of myometrial contractility, and that inappropriate synthesis of this mediator may trigger preterm delivery. In addition this study, by providing the first evidence that a NOS inhibitor is capable of initiating delivery in a rodent model, establishes a novel investigative platform from which further exploration of the functional role of the NO system in the pregnant myometrium may be launched.


    Notes
 
1 To whom correspondence should be addressed at: Sezione di Ostetricia e Ginecologia, Dipartimento di Medicina e Scienze dell'Invecchiamento, Facoltà di Medicina e Chirurgia, Università `G. d'Annunzio', Presidio Ospedaliero SS. Annunziata, Colle dell'Ara, 66100, Chieti, Italy. E-mail: tiboni{at}unich.it Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on March 13, 2000; accepted on May 22, 2000.