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Endocrinology Vol. 140, No. 10 4434-4441
Copyright © 1999 by The Endocrine Society

ARTICLES

Relaxin Up-Regulates the Nitric Oxide Biosynthetic Pathway in the Mouse Uterus: Involvement in the Inhibition of Myometrial Contractility1

Daniele Bani, Maria Caterina Baccari, Silvia Nistri, Franco Calamai, Mario Bigazzi and Tatiana Bani Sacchi

Departments of Anatomy, Histology and Forensic Medicine (section Histology) (D.B., S.N., T.B.S.) and Physiology (M.C.B., F.C.), University of Florence, and Prosperius Institute (M.B.), I-50139 Florence, Italy

Address all correspondence and requests for reprints to: Prof. Tatiana Bani Sacchi, Dipartimento di Anatomia, Istologia e Medicina Legale, Sezione di Istologia, Via le G. Pieraccini 6, I-50139 Florence, Italy. E-mail: histology{at}cesit1.unifi.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The uterus is a site of nitric oxide (NO) production and expresses NO synthases (NOS), which are up-regulated during pregnancy. NO induces uterine quiescence, which is deemed necessary for the maintenance of pregnancy. Relaxin is known to promote uterine quiescence. Relaxin has also been shown to stimulate NO production in several targets. In this study we investigated the effects of relaxin on the NO biosynthetic pathway of the mouse uterus. Estrogenized mice were treated with relaxin (2 µg) for 18 h, and the uterine horns were used for determination of immunoreactive endothelial-type NOS and inducible NOS. Moreover, uterine strips from estrogenized mice were placed in an organ bath, and the effect of relaxin on K+-induced contracture was evaluated in the presence or absence of the NOS inhibitor nitro-L-arginine. Relaxin increases the expression of endothelial-type NOS in surface epithelium, glands, endometrial stromal cells, and myometrium, leaving inducible NOS expression unaffected. Moreover, relaxin inhibits myometrial contractility, and this effect is blunted by nitro-L-arginine, thus indicating that the L-arginine-NO pathway is involved in the relaxant action of relaxin on the myometrium. Because relaxin is elevated during pregnancy, it is suggested that relaxin has a physiological role in the up-regulation of uterine NO biosynthesis during pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE NONPREGNANT state, the uterus contracts phasically and in response to stretch similarly to most organs provided with a smooth muscle coat. During pregnancy, the uterus undergoes major functional changes. In fact, it can distend and grow to accommodate the enlarging conceptus, yet remaining relatively quiescent until term. The mechanisms that enable the pregnant uterus to resist activation of contractility remain largely unknown. In recent years, the attention of researchers has been focused on the endogenous smooth muscle relaxant and second messenger nitric oxide (NO). NO is a free radical with a broad spectrum of biological activities (reviewed in Ref. 1) that is produced enzymatically from L-arginine by NO synthases (NOS). It has been repeatedly reported that NO can induce uterine relaxation (2, 3, 4, 5, 6, 7). Moreover, it has been shown that the mammalian uterus is a site of endogenous production of NO and that uterine tissues express NOS messenger RNA (mRNA) and protein (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). There is a general agreement that NO production and NOS expression are up-regulated during pregnancy and decline dramatically during labor at term concurrently with the onset of the coordinated uterine contractions required for delivery (8, 11, 12, 13, 14). The role of ovarian steroid hormones in the up-regulation of uterine NO production and NOS expression occurring during pregnancy is still unresolved. Studies in rats and rabbits have shown that estrogens inhibit NO generation by the uterus (19) and down-regulate NOS activity (20). On the other hand, progesterone, which is able to potentiate the relaxant action of NO on the myometrium, does not seem to influence per se the production of NO (19), even if antiprogestin drugs have been found to decrease mRNA for the inducible NOS isoform (iNOS) in the rat uterus (12).

Among the hormones called upon to promote uterine quiescence during pregnancy, relaxin (RLX) plays a prominent role. RLX is a peptide hormone, produced primarily by the corpus luteum of pregnancy, decidua, and trophoblast, which exerts multiple actions on the uterus, including the inhibition of myometrial contractile activity (reviewed in Refs. 21, 22). The mechanisms of action of RLX on the myometrium are multiple and remain to be fully clarified. They include a decrease in intracellular Ca2+ and an increase in cAMP, both leading to a reduction of myosin light chain kinase activity and, hence, to muscle cell relaxation as well as an activation of Ca2+-activated and ATP-sensitive K+ channels, resulting in hyperpolarization of the myometrial cell membrane (reviewed in Ref. 23). In recent years, several studies by our group have provided increasing evidence that RLX acts on several targets through stimulation of the endogenous production of NO (24, 25, 26, 27, 28, 29). Therefore, it is conceivable that the relaxant action of RLX on the uterus may also depend on its ability to up-regulate the NO biosynthetic pathway. The current study was designed to validate this hypothesis by investigating the effect of RLX on NOS expression and myometrial contractility in the presence of a NOS inhibitor in the mouse uterus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Eighteen sexually mature, virgin female mice of the Swiss albino strain, 8 weeks old and weighing about 30 g, were used. In previous studies, we found that uteri of mice with these characteristics were fairly responsive to RLX (30). The animals were purchased from a commercial dealer (Morini, Reggio Emilia, Italy), fed standard laboratory chow and water, available ad libitum, and housed under a 12-h light, 12-h dark photoperiod. The experimental protocol was designed in compliance with the Principles of Laboratory Animal Care (NIH Publication 86–23, revised 1985) and the recommendations of the European Economic Community (86/609/CEE), under supervision of a competent local committee for the care and use of laboratory animals.

Exp 1: evaluation of NOS expression
Twelve mice, chosen at random, received 5 µg 17ß-estradiol valerate (Progynon depot, Schering AG, Berlin, Germany). The hormone was dissolved in 0.5 ml sesame oil as repository vehicle and injected sc in a single dose. Estrogen treatment is required to induce uterine responsiveness to RLX (31, 32). The day of estradiol injection was taken as day 1 of the experiments. On day 6, the animals were randomly distributed into two groups of six mice each. The mice in the first group were treated with a sc injection of 2 µg highly purified porcine RLX (2500–3000 U/mg), prepared according to the method of Sherwood and O’Byrne (33). RLX was provided by Dr. O. D. Sherwood, University of Illinois (Urbana, IL). The hormone was dissolved in 0.5 ml 1% benzopurpurin (Fluka AG, Buchs, Switzerland) in PBS as a repository vehicle, which allows a slow release of the hormone over 24 h. The mice in the second group were injected with vehicle alone and used as controls. Eighteen hours after injection of RLX or vehicle, the mice were killed by neck elongation. Uterine horns were quickly removed and processed for immunohistochemical detection of NO synthases. To this end, the uterine horns were fixed by immersion in isotonic 4% paraformaldehyde in PBS, pH 7.4, dehydrated in graded ethanol, and embedded in paraffin wax. Two series of sections, 5 µm thick, were cut from the midportion of the uterine horns from both the control and RLX-treated mice. One series underwent immunostaining with a rabbit polyclonal antiserum specific for the calcium-dependent, endothelial-type NOS (eNOS) isoform (Alexis, Laufelfingen, Switzerland; raised against bovine endothelial eNOS; working dilution, 1:200); the other series was immunostained with a rabbit polyclonal antiserum specific for the calcium-independent, iNOS isoform (Alexis, raised against mouse macrophage iNOS; working dilution, 1:400). The antisera were diluted in Tris-buffered saline and applied to sections overnight at 4 C. Immune reaction was revealed by biotinylated goat antirabbit Igs, followed by streptavidin-conjugated alkaline phosphatase (DAKO Corp., Carpinteria, CA). Enzyme activity was revealed by incubation with naphtol As-Bi phosphate (Sigma Chemical Co., St. Louis, MO) as substrate and with New Fuchsin (Sigma Chemical Co.) as chromogen. Negative controls were carried out by omitting the primary antisera. Vascular endothelium and arterial smooth muscle were used as internal positive controls for eNOS and iNOS, respectively. When needed, sections were counterstained with Mayer’s hemalum. Counterstaining was not carried out on the sections used for computer-assisted densitometric analysis. This technique was used to quantify the intensity of the immunostaining and hence to achieve a quantitative evaluation of the expression of the two NOS isoforms. Measurements were performed according to the method described previously for similar purposes (24). From each uterus, five microscopic fields at a x1240 magnification were chosen at random from five different uterine tissue components, i.e. luminal epithelium, glands, endometrial stromal cells, and circular and longitudinal myometria. The fields were viewed by a CCTV television camera (Sony, Tokyo, Japan) applied to the light microscope and interfaced with an Apple Macintosh LC III personal computer through a Videospigot card (Supermac, Sunnyvale, CA). The card allows for the light transmitted across the microscopical slide to be determined within a range of 256 gray levels, which are comprised between 0 (black level) and 255 (white level). The card also allows for digitized images of the microscopic fields to be reproduced on the basis of the values estimated. The tissue areas to be analyzed were delineated, and other tissue components present in the microscopic field were deleted. Then, the OD of the immunostaining was measured using the free share Image analysis program, version 1.49 (NIH, Bethesda, MD).

Exp 2: evaluation of mechanical activity
Six mice were treated with 5 µg 17ß-estradiol valerate (Progynon depot, Schering AG) dissolved in 0.5 ml sesame oil as repository vehicle and injected sc in a single dose. Forty-eight hours later, the mice were killed by neck elongation. The uteri were removed carefully to avoid excessive stretching and placed on a petri dish containing Krebs solution at 37 C, and each uterine horn was cut longitudinally in two strips, 15 mm in length. The composition of the Krebs solution was as follows: NaCl, 118 mM; KCl, 4.5 mM; MgSO4, 1.0 mM; KH2PO4, 1.0 mM; NaHCO3, 25 mM; CaCl2, 2.5 mM; and glucose, 6.0 mM, gassed with a mixture of 95% O2 and 5% CO2. The mechanical activity of the uterine strips was recorded continuously under isometric conditions. One end of each uterine strip was tied to a platinum rod, and the other was connected by a silk thread to a force displacement transducer (FT03, Grass Instrument Co., Quincy, MA) coupled to a polygraph (model 7700, Sanborn, Waltham, MA). Each uterine strip was mounted in a 5-ml organ bath containing Krebs solution gassed with a 95% O2-5% CO2 mixture and kept at 37 C. Strips were allowed to equilibrate for 1 h under an initial load of 0.8 g. After equilibration, the uterine strips were activated with 35 mM K+ (as KCl). Only strips showing a stable response to the administered agonist were used in experiments (n = 10). When contractures reached a plateau, RLX was added to the bath medium to reach a final concentration of 10-8 M. After repeated washings with Krebs solution lasting for an overall period of at least 30 min, treatment with K+ and RLX was repeated to verify that the responsiveness of the uterine strips was not impaired with time. After subsequent 30-min washing, the NO synthase inhibitor NG-nitro-L-arginine (L-NNA; Sigma Chemical Co.) was added to the bath medium to reach a 10-3-M final concentration 15 min before induction of contractions with 35 mM K+. The concentration and exposure time of L-NNA used in our experiments were chosen because they have been previously shown to allow complete NOS inhibition (34). In fact, L-NNA is a competitive NOS inhibitor and only becomes effective when it overwhelms the natural substrate L-arginine present in the tissue. RLX was then added to the medium at a final concentration of 10-8 M in the continued presence of L-NNA to verify whether NOS inhibition could modify the response of the uterine strips to RLX. To exclude any possible influence of RLX on uterine nerve endings, some experiments were carried out in the presence of the nerve ending blocker tetrodotoxin (Sigma Chemical Co.) at a 10-6-M final concentration.

Relaxant responses are expressed as the percent decrease in the muscle tension induced by K+, which is assumed to be 100%.

Statistical analyses
The reported data are expressed as the mean ± SEM. Student’s t test for unpaired values was used for statistical analysis of the morphometrical data. Wilcoxon’s test was used for statistical analysis of the values obtained from measurements of uterine mechanic activity. Calculations were carried out using a GraphPad Prism 2.0 statistical program (GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NOS expression by uterine tissues
Immunostaining with anti-NOS antisera of the uteri from estrogenized control mice showed that the different uterine tissue components expressed both eNOS and iNOS at varying degrees. Upon visual examination, eNOS immunoreactivity was more intense in the luminal epithelium, glands, and longitudinal myometrium than in endometrial stromal cells and circular myometrium (Figs. 1Go and 2Go). Treatment with RLX of the estrogenized mice caused an increase in eNOS immunostaining, especially in the luminal epithelium (Fig. 1Go) and longitudinal myometrium (Fig. 2Go).



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  		Figure 1. Immunoreactivity for eNOS in the endometrium from mice treated with estrogen (left column) or estrogen plus RLX (right column). In the estrogen-treated mice, a weak immune reaction is visible in the endometrial tissues (a). At higher magnification, immunostaining is mainly appreciable in the supranuclear region of the cells of the luminal epithelium (b) and is homogeneously distributed in the cytoplasm of the glandular cells (c). RLX appears to enhance eNOS immunostaining in the endometrial tissues, especially in the luminal epithelium (d). At higher magnification, intense and homogeneous immunostaining can be seen in most of the cells of luminal epithelium (e) and in glandular cells (f). Nuclei are counterstained with Mayer’s hemalum. Magnification: a and d, x260; b, c, e, and f, x500. Bar, 20 µm.

 


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  		Figure 2. Immunoreactivity for eNOS in the myometrium from mice treated with estrogen (a) or estrogen plus RLX (b). In the estrogen-treated mice, a weak immune reaction is apparent in both the circular and longitudinal myometrium. RLX appears to enhance eNOS immunostaining in the myometrium, especially in the longitudinal layer. Of note, the immune reaction is always intense in the serosal epithelium. Nuclei are counterstained with Mayer’s hemalum. Magnification, x260. Bar, 20 µm.

 
Immunoreactive iNOS was also present in the different tissue components of the uteri from the estrogenized control mice. As for eNOS, iNOS immunostaining was more intense in the luminal epithelium, glands, and longitudinal myometrium than in endometrial stromal cells and circular myometrium (Figs. 3Go and 4Go). Treatment with RLX did not cause appreciable changes in iNOS immunoreactivity in any of the uterine tissue components (Figs. 3Go and 4Go).



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  		Figure 3. Immunoreactivity for iNOS in the endometrium from mice treated with estrogen (left column) or estrogen plus RLX (right column). In the estrogen-treated mice, an immune reaction is visible in the endometrial tissues (a). At higher magnification, immunostaining is mainly appreciable in the supranuclear region of the cells of the luminal epithelium (b) and is homogeneously distributed in the cytoplasm of the glandular cells (c). RLX does not have any appreciable effect on iNOS immunostaining (d). At higher magnification, the intensity and intracellular distribution of the immunostaining in cells of luminal (e) and glandular (f) epithelia are similar to those of mice treated with estrogen alone. Nuclei are counterstained with Mayer’s hemalum. Magnification, a and d, x260; b, c, e, and f, x500. Bar, 20 µm.

 


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  		Figure 4. Immunoreactivity for eNOS in the myometrium from mice treated with estrogen (a) or estrogen plus RLX (b). In the mice treated with estrogen alone, an immune reaction is apparent in both the circular and longitudinal myometria. RLX does not have any appreciable effect on iNOS immunostaining. Nuclei are counterstained with Mayer’s hemalum. Magnification, x260. Bar, 20 µm.

 
Computer-assisted densitometry showed that RLX treatment significantly increased eNOS expression in all uterine tissue components, especially the luminal epithelium and longitudinal myometrium (Fig. 5AGo). On the other hand, RLX treatment had no significant effect on iNOS expression by the uterus (Fig. 5BGo).



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  		Figure 5. Densitometric analysis of the immunostaining for eNOS (A) and iNOS (B) in uterine tissues from mice treated with estrogen (open columns) or estrogen plus RLX (dotted columns). RLX increases significantly the OD of eNOS immunostaining in all uterine tissue components. No significant changes were evident in the OD of iNOS immunostaining in any uterine tissue components after RLX treatment. For each group, n = 6. Differences were not significant unless otherwise indicated: *, P < 0.02; **, P < 0.01; ***, P < 0.0005; ****, P < 0.0001.

 
Uterine contractility
After 1 h of equilibration, the uterine strips showed spontaneous, irregular contractile activity. Exposure of the uterine strips to 35 mM K+ produced a sudden rise in tension that reached a plateau within 1 min (mean amplitude, 1.9 ± 0.2 g). In 5 of 10 preparations examined, K+-induced contracture was associated with the disappearance of any sign of spontaneous muscle activity. The plateau phase was maintained for the whole period of exposure to K+ (observed up to 15 min). Upon washout, strip tension returned to baseline.

RLX, added to the bath medium during K+-induced contracture, caused a slowly developing, sustained relaxant response with a latency of 12–15 sec (mean percent decrease in K+-induced tension, 55.9 ± 3%; Fig. 6Go). Relaxation was maintained for the overall observation period (10 min). A subsequent application of RLX, carried out 30 min later and after repeated and prolonged washes with Krebs solution, produced a response similar to that to the former application, thus indicating that no desensitization to the hormone occurred. The relaxant response of the uterine strips to relaxin was not due to stimulation of uterine nerves, because it was not influenced by the nerve ending blocker tetrodotoxin.



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  		Figure 6. Representative recordings of the effect of RLX on K+-induced contracture of an uterine strip from an estrogenized mouse in vitro, obtained in the absence (left) and presence (right) of the NOS inhibitor L-NNA. The amplitude of the relaxant response of the contracted myometrium to RLX is substantially reduced by L-NNA. The two traces illustrate two consecutive experiments performed on the same strip.

 
Incubation of uterine strips (n = 5) with the NO synthase inhibitor L-NNA, added to the bath medium 15 min before treatment with K+ and RLX, resulted in a clear-cut, significant reduction of the relaxant effect of RLX treatment (Fig. 6Go); the mean percent decrease in K+-induced tension was 21.4 ± 2.4% vs. 55.9 ± 3% found in the absence of L-NNA (P < 0.05). Of note, L-NNA per se did not cause substantial changes in the myometrial contractility pattern, either spontaneous or stimulated by K+.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study show that RLX, administered systemically to estrogenized mice, increases the expression of eNOS by uterine tissues, thus indicating that this hormone is a regulatory agent for the production of NO by the uterus. Taking into account that RLX is elevated during pregnancy (35, 36, 37), the present findings suggest that RLX may be responsible for up-regulation of NO biosynthesis, which occurs in the uterus during pregnancy (8, 11, 12, 13, 14) and is deemed necessary to favor myometrial quiescence aimed at facilitating accommodation of the conceptus. In this study, we also provide the first experimental evidence that the inhibitory action of RLX on uterine contractility involves the endogenous generation of NO. In fact, the relaxant effect of RLX on K+-precontracted uterine strips is significantly blunted by the NOS inhibitor L-NNA. Our findings are in contrast with those of a previous study (38), in which the relaxant effect of RLX on isolated rat uteri stimulated by oxytocin was not antagonized by the NOS inhibitor L-NAME (10-5 M). This result may depend on the use of too low a concentration of L-NAME, about 100-fold lower than the concentration of L-NNA used by us, as well as on lower potency of L-NAME as a NOS inhibitor compared with L-NNA (39). Despite the fact that we used L-NNA at high concentrations, blockade of the L-arginine-NO pathway did not result in a complete inhibition of the myometrial response to RLX. This is not surprising, because RLX is known to act as a relaxant for the myometrium through multiple mechanisms (reviewed in Ref. 23), some of which are independent of NO. Indeed, the redundance of regulatory mechanisms for maintaining myometrial quiescence may be needed to prevent premature evolution of parturient activity.

Interestingly, the current findings indicate that RLX up-regulates constitutive eNOS, but leaves inducible iNOS expression unaffected. It is known that the activity of eNOS is regulated acutely by changes in the intracellular Ca2+ concentration (40) and, therefore, can be quickly modulated, whereas iNOS is transcriptionally regulated (41, 42) and requires much longer times than eNOS to be functionally modulated. It is conceivable that the RLX-induced generation of NO in uterine tissues, which is sustained by eNOS, may follow closely the trend of RLX production, which is elevated during pregnancy and declines abruptly before labor (35, 36, 37). Moreover, studies in rats have shown that uterine iNOS mRNA, which also increases during pregnancy, already drops in the preterm period and decreases further during labor (12). One might speculate that RLX is required to contribute needed NO when iNOS-derived NO is reduced, thus allowing the NO-dependent inhibition of the contractile activity of the myometrium to persist until the onset of labor. At labor, concurrently with the decline in circulating RLX, the drop in eNOS-derived NO could therefore remove irresponsiveness of the uterus to contraction-inducing agents and, hence, initiate delivery.

The present findings show that not only the myometrium but other uterine tissue components also express NOS. This is in agreement with previous studies in which NOS expression has been reported in luminal epithelium (9, 14, 15, 16, 18), glandular epithelium (9, 10, 14, 15, 16), and endometrial stromal cells (10, 15, 16). We have found that RLX increases eNOS expression at all of these endometrial sites as it does in the myometrium. The physiological meaning of the RLX-induced up-regulation of eNOS expression in endometrial epithelial and stromal cells remains to be clarified. Besides an autocrine role in regulating specific cell functions, as in the myometrium, it is possible that NO generated by the endometrial tissues may also have a paracrine role in influencing the function of tissues nearby. In fact, NO is a highly diffusible gas that can easily reach cells and tissues somewhat distant from the site of production, as occurs in blood vessels in which NO produced by the endothelium induces relaxation of the smooth muscle cell coat (43). It could be hypothesized that NO produced by the endometrium under the influence of RLX may contribute to a moment to moment regulation of the myometrial quiescence, with a preferential action on the circular myometrium, which is contiguous to the endometrium and expresses lower levels of immunoreactive eNOS and iNOS than the longitudinal myometrium. The difference in NOS expression between the two myometrial layers observed by us agrees with the results of previous studies showing that the two muscle layers of the rodent uterus have different physiological and pharmacological characteristics (44, 45, 46, 47, 48, 49), including different responses to the relaxant actions of progesterone (49) and RLX (38).


   Acknowledgments
 
The authors gratefully acknowledge Dr. O. D. Sherwood from the Department of Molecular and Integrative Physiology, University of Illinois at Urbana Champaign (Urbana, IL), for having provided purified porcine relaxin as a gift. Many thanks are also due to Ms. Laura Calosi and Mr. Daniele Guasti, Department of Anatomy, Histology, and Forensic Medicine, University of Florence (Florence, Italy), for skillful technical assistance.


   Footnotes
 
1 This work was supported by government funds from the Italian National Research Council and the Italian Ministry of the University (MURST, funds 40% and 60%), Rome, Italy. Back

Received March 3, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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