Effects of L-arginine and sodium nitroprusside on the spontaneous contractility of human non-pregnant uterus

P. Hoffmann1, F. Stanke-Labesque2, R. Fanchin3, N. Dilaï1, J.C. Pons1 and J.M. Ayoubi1

1 Department of Obstetrics and Gynaecology and 2 Laboratory of Pharmacology, Grenoble University Hospital, Grenoble, BP219, 38043 Grenoble, Cedex 9 and 3 Department of obstetrics and Gynaecology and Reproductive Endocrinology, Hospital Antoine Béclère, Clamart, France 4 To whom correspondence should be addressed. e-mail: PHoffmann{at}chu-grenoble.fr


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The aim of this study was to investigate, in isolated human non-gravid myometrium, the involvement of the cyclic guanosine monophosphate (cGMP) pathway in nitric oxide (NO) induced relaxation. METHODS: Strips of human myometrium from hysterectomized women were suspended in organ baths for recording of isometric tension. Cumulative concentration-response curves for L-arginine and sodium nitroprusside were performed in the presence of methylene blue (10 µmol/l) or vehicle (control). The effect of increasing concentrations of 8-bromo-cGMP on uterine spontaneous contraction was also studied. RESULTS: L-arginine and sodium nitroprusside induced a concentration-dependent decrease in the amplitude of the myometrial spontaneous contractions. Pre-treatment with methylene blue enhanced the inhibitory effect of L-arginine and sodium nitroprusside on myometrial spontaneous contractions. In addition, 8-bromo-cGMP had no effect on spontaneous contractions in human myometrium. CONCLUSIONS: These data provide evidence that L-arginine and sodium nitroprusside inhibit the spontaneous contractions of the non-pregnant human uterus through a cGMP independent pathway.

Key words: cGMP pathway/human non-pregnant uterus/methylene blue/nitric oxide


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nitric oxide (NO) has been found to be of great importance throughout the body as a vasodilator, neurotransmitter and relaxant of vascular and non-vascular smooth muscle. It has a determinant role in the control of uterine contractility. Endogenous NO is produced from uterine smooth muscle cells, from its precursor L-arginine, via NO synthases (NOS). There have been only a few studies examining the regulatory function of NO in the non-pregnant uterus (Telfer et al., 1995Go). However, evidence demonstrates that there is an inverse correlation between the frequency of uterine contractions and the pregnancy rate (Fanchin et al., 1998Go). Furthermore, clinical trials have shown a positive effect of the NO-precursor L-arginine, on the pregnancy rate of poor responder patients (Battaglia et al., 1999Go). Kuenzli et al. (1998Go) investigated the existence of functionally relevant NOS in the non-pregnant monkey uterus. They suggested a role for NO in the moment-to-moment regulation of monkey uterine contractile activity, and showed that if guanylyl cyclase served as a target for NO, activation of this enzyme was not required for NO-dependent uterine relaxation.

The aim of our study was to investigate the existence of such functionally relevant NOS in the human non-pregnant uterus, and to explore the NO-dependent relaxation pathway.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Chemicals
Drugs and their sources were: KCl (Prolabo Normapur, France), L-arginine, sodium nitroprusside, methylene blue, Nw-Nitro-L-arginine, 8 bromo-cGMP from Sigma (L’Isle d’Abeau, France). Stock solutions were prepared in distilled water. All drugs were stored at –20°C and freshly dissolved in distilled water to the appropriate molar concentration in the organ bath.

Tissue preparation
Uterus strips were harvested from 18 women aged 47–52 years, undergoing hysterectomy for dysfunctional uterine bleeding. This investigation conforms to the principles outlined in the Declaration of Helsinki and was approved by the medical ethics committee of the Academic Medical Hospital. None of the women had any hormonal treatment for at least 1 month prior to their hysterectomy and none had previously been treated with GnRH analogues.

The strips were removed in the Calza’s fasciculus (i.e. in the anterior uterine wall midway between the internal os and the fundus), leaving at least 3 mm of myometrium on both the endometrial and serosal margins in order to use the stratum vasculare. They were immediately placed in Kreb’s solution maintained at 4°C, and transported to the laboratory where they were stored at 4°C and used within 24 h. The Kreb’s solution had the following composition: NaCl (118.0 mmol/l), KCl (4.7 mmol/l), CaCl2 (2.5 mmol/l), MgSO4 (1.0 mmol/l), KH2PO4 (1.0 mmol/l), glucose (11.0 mmol/l) and NaHCO3 (25.0 mmol/l).

Measurement of isometric tension in human uterine strips
The strips were cut into 2.5x2.5x15.0 mm pieces (Bradley et al., 1998Go), mounted on parallel wires and placed in a 10 ml organ bath filled with Krebs solution maintained at 37°C and gassed with a mixture of 5% CO2, 95% O2. The lower wire was fixed to a micrometer (Mitutoyo, Japan) and the upper wire was attached to a force transducer (UF-1; Pioden, UK) to allow changes in tension to be recorded isometrically. Changes in tension were monitored on a Linseis recorder (Bioblock, France). Each strip of myometrium was set up under an initial tension of 2 g as previously described (Buhimshi et al., 1995Go; Kotrzevska et al., 1997Go; Bradley et al., 1998Go) and allowed to equilibrate for 60 min; the Kreb’s solution was changed every 15 min. The strips were then challenged once by the addition of 90 mmol/l KCl. After a 120 min stabilization period, strips were stabilized and developed a regular and phasic contracting activity.

Cumulative concentration-response curves for L-arginine (log increments, 1 µmol/l to 1 mmol/l, every 10 min) and sodium nitroprusside (SNP) (log increments, 10 nmol/l to 0.1 mmol/l, every 10 min) were performed. The involvement of endogenous NO in L-arginine mediated relaxation was assessed by prior incubation with the specific NO synthase inhibitor Nw-Nitro-L-arginine (L-NA) 100 µmol/l (Musch and Busse, 1990Go). The involvement of the guanylyl cyclase pathway was assessed by prior incubation with methylene blue (10 µmol/l) for 30 min. In addition, cumulative concentration-response curves to the cyclic GMP analogue 8-bromo-cGMP (log increments, 10 nmol/l to 0.1 µmol/l, every 10 min) were also performed. As far as possible, experiments were performed with strips from the same uterus. Appropriate controls (incubation with solvents) were run under similar experimental conditions in rings of uterus obtained from the same woman. Only one concentration-response curve was performed in each uterine strip. All experiments were conducted with aluminium foil-covered organ baths to prevent light-induced degradation of the drugs.

Statistical analysis
Contractile responses were expressed as a percentage of the contraction induced by KCl (90 mmol/l). The concentration of relaxing agent producing 50% of the maximal effect (EC50) was determined from each curve by a logistic curve fitting equation. The pD2 (potency) represents the negative logarithm of the EC50. Results are expressed as mean ± SEM for the specified number of samples tested. Analysis of variance (ANOVA) for repeated measures was performed, followed by the Bonferroni corrected t-test. Individual comparisons were made by Student’s t-test for paired data. P-value of <0.05 was considered to be significant.


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 Materials and methods
 Results
 Discussion
 References
 
Effects of L-arginine on uterine spontaneous contractions
Human myometrial strips from non-pregnant women developed spontaneous phasic contractile activity (Figure 1).



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Figure 1. Typical contractile activity of human uterine tissue strips representative of results obtained after a 2 h period of stabilization.

 
The addition of increasing concentrations of L-arginine to contractile myometrial strips induced a concentration-dependent partial inhibition of the phasic contractile uterine activity (Figure 2). The spontaneous contraction was reduced by 65% in the presence of L-arginine 0.1 mmol/l (Table I).



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Figure 2. Cumulative concentration-response curve of L-arginine on spontaneous contraction in the presence of methylene blue (10 µmol/l), NW-nitro-L-arginine (L-NA) (100 µmol/l) or vehicle (control). Results are expressed as a percentage of inhibition of spontaneous contractility and are presented as means ± SEM of five experiments.

 

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Table I. Potency (pD2) and efficacy (Emax expressed as percentage of inhibition of uterine contractility) of L-arginine and sodium nitroprusside (SNP) with or without methylene blue pre-treatment, and of L-arginine with NW-nitro-L-arginine (L-NA) pre-treatment. n indicates the number of strips. Results are presented as mean ± SEM of n experiments
 
Pre-treatment with methylene blue, which by itself did not change uterine contractility, induced no significant change in L-arginine-induced relaxation of uterine strips (Table I, Figure 2).

In addition, L-NA significantly inhibited the L-arginine-induced relaxation (P = 0.016 versus control) (Table I, Figure 2).

Effect of SNP on uterine spontaneous contractions
The addition of increasing concentrations of SNP induced a concentration-dependent inhibition of the phasic contractile activity of human myometrial strips (Figure 3). The spontaneous contraction was reduced by 92% in the presence of 0.1 mmol/l SNP (Table I).



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Figure 3. Cumulative concentration-response curve of SNP on spontaneous contraction in the presence of methylene blue (10 µmol/l) or vehicle (control). Results are expressed as percentage of inhibition of spontaneous contractility and are presented as means ± SEM of six experiments.

 
Pre-treatment with methylene blue did not prevent SNP-induced relaxation of uterine strips (Figure 3). In contrast, in terms of potency, pre-treatment with methylene blue induced a significant potentiation of SNP (Table I).

Effect of 8 Bromo-cGMP on uterine spontaneous contractions.
The addition of increasing concentrations of 8 Bromo-cGMP to contractile myometrial strips had no significant effect on myometrium spontaneous contractions.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study demonstrated, on human non-pregnant uterus, that the NO precursor L-arginine and the NO donor SNP both inhibited spontaneous contractions in vitro through a cGMP-independent pathway.

NO is produced from the conversion of L-arginine to L-citrulline by a family of enzymes known as NOS. The functional observation that L-arginine had the capability of relaxing in vitro the spontaneous contractions of myometrial strips from non-pregnant uterus suggests the presence of functional NOS in non-pregnant uterus. The relaxant effect of L-arginine has previously been demonstrated in pregnant myometrium from human and rats (Izumi et al., 1993Go). Compelling evidence for the presence of NOS has been provided by immunocytochemistry and in-situ hybridization of NOS mRNA in uterus from pre- and post-menopausal women (Telfer et al., 1995Go) or by Western immunoblot analysis (Khorram et al., 1999Go), as well as by biochemical analysis of NOS activity in both pregnant and non-pregnant women (Ramsay et al., 1996Go). Our results confirm the presence of a functional NOS in human non-pregnant myometrium, since the NOS substrate L-arginine induced a dose-dependent inhibition of the uterine spontaneous contractile activity. These data are consistent with those of Buhimschi (1995Go) who demonstrated that L-arginine but not D-arginine decreased uterine spontaneous contractility in human uterus. Moreover, the inhibitory effect of L-NA on L-arginine-evoked uterine relaxation confirms the role of NO in L-arginine-mediated relaxation.

Similarly, the NO donor SNP inhibited spontaneous contraction in non-pregnant uterus (present study). On guinea pig myometrium the NO donor S-nitroso-L-cysteine (CysNO) had no effect on spontaneous contractile activity, either in pregnant or non-pregnant uterine tissue strips, but had a relaxing activity on agonist-evoked contractions (Kuenzli et al., 1996Go). These data suggest the variability of the effect of NO donors according to the species studied and the experimental conditions. Indeed, according to the onto- and phylogenetical differences in the myometrium layers, the expression of estrogen and progesterone receptors depends on the tissue studied (Noe et al., 1999Go). The presence of a NO–cGMP dependent pathway in the human uterus was first investigated during pregnancy by Buhimschi (1995Go), who proposed that this pathway was responsible for maintaining uterine quiescence during pregnancy. However, the inhibitory effect of CysNO on uterine contraction has been reported to be independent of the cGMP pathway in both guinea pig (Kuenzli et al., 1996Go) and non-pregnant monkey myometrium (Kuenzli et al., 1998Go). In line with these findings, our results showed that pre-treatment with methylene blue did not reduce L-arginine and SNP-induced inhibition of spontaneous uterine contractions. These data suggest that L-arginine and SNP inhibited the in-vitro spontaneous contractions of non-pregnant human myometrium through a cGMP-independent pathway. Conversely, the production of cGMP in uterus after an L-arginine or NO-donor challenge is well established. Indeed, an increased production of cGMP in response to L-arginine (Buhimschi et al., 1995Go; Buxton et al., 2001Go), diethylamine/NO (Buhimschi et al., 1995Go) or CysNO (Buxton et al., 2001Go) has been reported in human non-pregnant uterus. Therefore, the production of cGMP after stimulation by L-arginine and SNP were not measured in the present study. In this context, we examined the effect of the stable cGMP analogue, 8 Br-cGMP, on myometrial strips from non-pregnant uterus. The observation that 8 Br-cGMP did not change myometrial spontaneous contraction suggests that NO-mediated inhibition of non-pregnant human spontaneous contraction is independent of the cGMP pathway. In human myometrium from non-pregnant women, our conclusions are consistent with those of Word et al. (1991Go) and Buxton et al. (2001Go). However, data on animal (Diamond, 1983Go; Izumi et al., 1993Go; Yallampalli et al., 1993Go; Kuenzli et al., 1996Go; Hennan and Diamond, 1998Go) and human (Norman and Cameron, 1996Go) pregnant uterus are still controversial.

Interestingly, guanylate cyclase blockade with methylene blue enhanced SNP-induced relaxation, but induced no significant change of L-arginine-induced relaxation (present study). These data are not easy to explain. However, K+ channels are known to be modulators of human myometrial contractile activity (Anwer et al., 1993Go; Cheuk et al., 1993Go; Morisson et al., 1993Go; Modzelewska et al., 1998Go). In addition, large conductance calcium-activated potassium channels (BKCa) modulate the vascular response to NO (Khan et al., 1997Go; Bang et al., 1999Go). Moreover, it has been reported that methylene blue directly activates calcium-dependent potassium channels in human vascular smooth muscle (Stockand and Samson, 1996Go). Therefore, it could be hypothesized that BKCa channels contribute to SNP-induced myometrial relaxation. Consistent with this hypothesis, Bradley demonstrated that CysNO-induced relaxation was completely reversed by a calcium-dependent potassium channel blocker (Bradley et al., 1998Go). In addition, it has been suggested that K+ATP channels could also be involved in diethylamine/NO-induced inhibition of spontaneous contractile activity of the non-pregnant human myometrium (Modzelewska et al., 1998Go). Other studies are, however, required to further explore the role of K+ channels in the modulation of NO-mediated relaxation in human non-pregnant myometrium.

In conclusion, the present study provides evidence that in human non-pregnant uterus L-arginine and SNP inhibit spontaneous uterine contraction through a cGMP-independent pathway. With regard to the variability of the effect of L-arginine and NO donors according to the species (Yallampalli et al., 1994Go; Buhimschi et al., 1995Go; Izumi and Garfield, 1995Go), the experimental conditions, and the hormonal status (Ramsay et al., 1996Go; Noe et al., 1999Go; Weeks et al., 1999Go; Battaglia et al., 2002Go), these data highlight the need for more experiments on human uterine preparations. However, further experiments are required to better understand the modulation of myometrium contractility. This would spur further investigation regarding the possibility for using an exogenous NO donor in IVF. Indeed structural abnormalities of the uterine wall in infertile women have been reported (Kunz et al., 2000Go) and should be responsible for abnormal contractile status. These data could open the field to the intra-vaginal administration of L-arginine in the very early stages of embryo transfer in order to improve implantation rates.


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 Materials and methods
 Results
 Discussion
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Submitted on July 1, 2002; accepted on October 2, 2002



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