Sezione di Ostetricia e Ginecologia, Dipartimento di Medicina e Scienze dell'Invecchiamento, Facoltà di Medicina e Chirurgia, Università `G. d'Annunzio', Chieti, Italy
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Abstract |
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Key words: L-NAME/mouse /nitric oxide/preterm delivery
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Introduction |
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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., 1995). As is well established, NOS activity can be competitively inhibited by a variety of L-arginine analogues (Moncada et al., 1995
). NO has been recently postulated as having a role in the complex molecular interplay which regulates myometrial function during gestation (Norman, 1996
; Sladek et al, 1997
; Yallampalli et al., 1998
), 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., 1993; Izumi et al., 1993
; Natuzzi et al., 1993
; Yallampalli et al., 1993
, 1994
), guinea-pig (Weiner et al., 1994
), rabbit (Sladeck et al., 1993), sheep (Figueroa and Massmann, 1995
) and mouse (Huang et al., 1995
; Moorhead et al., 1995
). In the rabbit (Sladeck et al., 1993) and rat (Yallampalli et al., 1993
), 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., 1993
; Izumi et al., 1993
). The responsiveness of the rat uterus to the relaxant effect of NO was found to be decreased during labour (Yallampalli et al., 1993
; Buhimschi et al., 1995b
).
Studies on the human pregnant uterus have documented myometrial NOS activity (Telfer et al., 1995; Ramsey et al., 1996; Thomson et al., 1997
; Bansal et al., 1997
; Ekerhovd et al., 1999
). The ability of exogenous NO to evoke myometrial relaxation has also been determined (DeSimone et al., 1990
; Greenspoon and Kovacic, 1991
; Buhimschi et al., 1995a
; Lee and Chang, 1995
). 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, 1997
; Ekerhovd et al., 1999
). A decline in human myometrial NOS expression in association with labour and delivery has been reported (Bansal et al., 1997
).
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, 1996; Sladek et al., 1997
; Yallampalli et al., 1998
). 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.
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Materials and methods |
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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 24 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.
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Results |
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Study 2
As illustrated in Figure 2, three of eight (37.5%) animals implanted with vehicle-filled pumps delivered preterm. In all three instances, gestations ended on GD 16.517 (Table II
). 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 2
). No clinical signs of maternotoxicity were observed in animals used in this study.
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Discussion |
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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., 1997). This NO donor has been found, albeit with some inconsistencies, to effect relaxation of pregnant myometrium (Sladek et al., 1997
). 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, 1990
) 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., 1995
; Moorhead et al., 1995
), and that uterine horns of pregnant mice exhibited isometric tension increments after application of a NOS inhibitor (Boquet et al., 1998
).
Over the last decade, a number of investigations (Yallampalli and Garfield, 1993; Diket et al., 1994; Molnar et al., 1994
; Buhimschi et al., 1995b
; Salas et al., 1995
; Helmbrecht et al., 1996
; Yallampalli et al., 1996
) 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., 1994
; Buhimschi et al. 1995b
; Helmbrecht et al., 1996
; Yallampalli et al., 1996
) or without (Diket et al., 1994
) 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., 1996
). 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., 1996
). 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., 1996
; Chwalisz et al., 1997
; Tschugguel et al., 1999
) 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 1418 (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.
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Notes |
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References |
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Submitted on March 13, 2000; accepted on May 22, 2000.