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Biology of Reproduction 66, 778-784 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Relaxin Depresses Small Bowel Motility Through a Nitric Oxide-Mediated Mechanism. Studies in Mice1

Daniele Bani2,a, Maria Caterina Baccarib, Silvia Quattronea, Silvia Nistria, Franco Calamaib, Mario Bigazzic, and Tatiana Bani Sacchia

a Departments of Anatomy, Histology, and Forensic Medicine, and b Physiological Sciences, c University of Florence, and Prosperius Institute, Florence, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gastrointestinal motility is reduced and the incidence of functional gastrointestinal disorders is increased in pregnancy, possibly due to hormonal influences. This study aims to clarify whether the hormone relaxin, which attains high circulating levels during pregnancy and has a nitric oxide-mediated relaxant action on vascular and uterine smooth muscle, also reduces bowel motility and, if it does, whether nitric oxide is involved. Female mice in proestrous or estrous were treated for 18 h with relaxin (1 µg s.c.) or vehicle (controls). Isolated ileal preparations from both groups were used to record contractile activity, either basal or after acute administration of relaxin (5 x 10-8 M). Drugs inhibiting nitric oxide biosynthesis or neurotransmission were used in combination with relaxin. Expression of nitric oxide synthase isoforms by the ileum was assessed by immunocytochemistry and Western blot analysis. Relaxin caused a clear-cut decay of muscle tension and a reduction in amplitude of spontaneous contractions upon either chronic administration to mice or acute addition to isolated ileal preparations. These effects were significantly blunted by NG-nitro-L-arginine, but not by the neural blockers we used. Moreover, relaxin increased the expression of nitric oxide synthases II and III, but not synthase I. Relaxin markedly inhibits ileal motility in mice by exerting a direct action on smooth muscle through the activation of intrinsic nitric oxide biosynthesis.

nitric oxide, relaxin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The concept that sex hormones play roles that are limited to reproductive functions is rapidly changing. For example, evidence is mounting that sex hormones may deeply influence gastrointestinal motility. In fact, intestinal transit of nutrients is slower during the luteal phase of the ovarian cycle than during the follicular phase [1] and, during pregnancy, the incidence of constipation increases [2] and gastrointestinal motility is reduced [3–5]. Moreover, because small bowel transit is slowed during gestation and rapidly returns to levels of the follicular phase in the postpartum period, strongly suggests that the above pregnancy-induced changes are hormonally related [4–6]. However, the issue of which pregnancy hormones are involved in these changes is controversial. Although studies in laboratory mammals have indicated progesterone as the predominant factor in the inhibition of gastrointestinal motility [7–13], this has not been confirmed in more recent studies in which administration of progesterone to ovariectomized rats has been shown to enhance gastric emptying, whereas it was estradiol, either alone or in combination with progesterone, that led to a slowing of gastric emptying [14].

Clinical observations also suggest an involvement of sex hormones in patients with unexplained gastrointestinal symptoms grouped under the disorders known as functional bowel diseases. Recently, a predominant gender expression in functional bowel diseases has been reported, with women outnumbering men by as much as 20:1 [15]. This striking disparity in gender expression may be explained by reproductive physiology, especially considering that women with functional bowel disease have a history of cycling gastrointestinal symptoms, which characteristically occur or worsen during the postovulatory phase of the menstrual cycle, when hormones are secreted by the developing corpus luteum [16].

In addition to ovarian steroids, the peptide hormone relaxin should also be considered as having the potential of influencing gastrointestinal motility. Relaxin is produced by the corpus luteum and attains high circulating levels during the luteal phase of the menstrual cycle [17] and, even more, during pregnancy [18, 19]. Unlike ovarian hormones, relaxin has not attracted the attention of researchers as a possible regulator of gastrointestinal motility. Thus, early reports that a relaxin preparation extracted from the ovary reduced the strength and frequency of contractions in the rat ileum [20], and that relaxin has disruptive effects on the migrating myoelectric complex of the rat small intestine [21] have remained isolated and unconfirmed.

The present study is designed to verify the hypothesis that relaxin is involved in the control of intestinal motility. This possibility relies on the following data: Relaxin has a relaxant action on the smooth muscle of several anatomical sites [22–24], specific relaxin-binding sites have been identified in smooth muscle cells of the small intestine in pregnant pigs [25], and high levels of circulating relaxin have been detected in 68% of 28 nonpregnant women with functional bowel disease [26].

We have previously demonstrated that relaxin exerts its relaxant action on smooth muscle cells by stimulating the endogenous production of nitric oxide (NO) [23, 24]. In turn, NO is known to mediate relaxation of gastrointestinal smooth muscle [27–29] and to be involved in pregnancy-associated decrease of gastrointestinal motility [30]. Moreover, the expression of NO synthase (NOS) is increased in gastrointestinal tissues during pregnancy [31, 32]. Therefore, a further aim of this study is to clarify whether relaxin may act on intestinal motility by influencing the NO biosynthetic pathway in the components of the intestinal wall, as it does in other target organs with smooth muscle [23, 24, 33].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Studies were carried out on 8- to 12-wk-old albino female mice of the Swiss strain, weighing about 30 g (Morini, Reggio Emilia, Italy). The mice were fed standard laboratory chow and water, and were housed under a 12L:12D photoperiod. The experimental protocol was designed in compliance with the Principles of Laboratory Animal Care (National Institutes of Health publication 86-23, revised 1985) and recommendations of the European Economic Community (86/609/CEE), under supervision of a competent local committee for the care and use of laboratory animals. After 1 wk of acclimatization, the mice underwent assessment of the phase of the estrous cycle by light microscopic examination of vaginal smears stained with Papanicolaou, according to the method of Austin and Rowlands [34]. Only mice in proestrous or estrous (i.e., the estrogen-dominated phases of the ovarian cycle) entered the experiments. This choice was made because estrogen is known to favor relaxin responsiveness of several target organs and tissues [35, 36]. The mice were randomly distributed to 2 groups of 6 animals each. Mice in the first group received a single s.c. injection of 1 µg of highly purified porcine relaxin (2500–3000 U/mg), generously donated by Dr. O.D. Sherwood, and prepared according to the method of Sherwood and O'Byrne [37]. The hormone was dissolved in 0.2 ml of 1% benzopurpurin (Fluka AG, Buchs, Switzerland) in PBS, a repository vehicle that allows a slow release of the hormone over 24 h. The chosen dose, vehicle, and route of administration of relaxin were similar or even lower than those used in previous in vivo studies in mice, and proved effective in inducing mammary gland growth [35] and up-regulating myometrial nitric oxide synthase (NOS) expression [24]. The mice of the second group received the vehicle alone and were used as controls. Eighteen hours later, the mice were killed by cervical dislocation. The abdomen was immediately opened and the ileum was removed and transversely cut into segments.

Functional Studies on Ileal Contractile Activity

For the functional studies, distal segments of the ileum were used. They were placed in Krebs-Henseleit solution, which consisted of 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 2.5 mM CaCl2, and 10 mM glucose pH 7.4, and bubbled with 95% O2/5% CO2. The lumenal content was gently flushed out. Whole full-thickness ileal preparations were placed longitudinally in 5-ml organ baths containing Krebs-Henseleit solution gassed with 95% O2/5% CO2, while the temperature was maintained within a range of 37 ± 0.5°C. Continuous recording of isometric tension was achieved by a Grass (Quincy, MA) FT03 force displacement transducer coupled to a Sanborn (Waltham, MA) 7700 polygraph. The preparations were allowed to equilibrate for at least 1 h under an initial load of 1–1.5 g. During this period, the preparations underwent repeated and prolonged washes with Krebs-Henseleit solution to avoid accumulation of metabolites in the organ baths.

A first series of data was obtained by evaluating the effects of addition of relaxin to the bath medium (5 x 10-8 M final concentration) of ileal preparations coming from either control mice or mice treated long-term (18 h) with relaxin. This relaxin concentration was used because it is in the range in which the contractile activity of the mouse uterus is effectively inhibited in vitro [24]. The hormone was given alone or in combination with the following drugs: the NOS inhibitor NG-nitro-L-arginine (L-NNA, 1 x 10-3 M), the nerve-ending blocker tetrodotoxin (TTX, 1 x 10-6 M), the muscarinic receptor antagonist atropine sulfate (1 x 10-6 M), and the N-type channel blocker {omega}-conotoxin GVIA (1 x 10-6 M) [38]. The reported concentrations were the final bath concentrations. All the drugs were obtained from Sigma Chemical Company (St. Louis, MO).

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 [24, 39]. In some experiments, L-NNA was added 30 min before challenging the organ baths with relaxin. Because L-NNA is a competitive inhibitor of NOS, a 30-min pretreatment was aimed at affording the inhibitor enough time to overcome the natural NOS substrate L-arginine and to completely inactivate NOS at the time of relaxin addition.

A second series of data was obtained by recording the basal contractile activity in the control mice and the mice under the chronic effect of 1 µg relaxin given systemically 18 h before they were killed.

Evaluation of NOS Expression by Immunohistochemistry

Ileal fragments from control mice and relaxin-treated mice were fixed by immersion in 4% paraformaldehyde in PBS pH 7.4 for 2 h, cryoprotected by incubation in 20% sucrose in PBS for 1 h, washed in PBS, and quickly frozen at -80°C in cryostat embedding medium (Bio-Optica, Milan, Italy). Cryostat sections, 6 µm thick, were cut from each ileal fragment and immunostained with rabbit polyclonal antibodies to reveal the different NOS isoforms: the constitutive, endothelial-type NOS (eNOS, or NOS III), the inducible NOS (iNOS, or NOS II), or the constitutive, neuronal-type NOS (nNOS, or NOS I). The polyclonal antisera used were from Calbiochem (San Diego, CA) and were applied to sections at working dilutions of 1:50 (anti-NOS I and III) or 1:100 (anti-NOS II) in PBS overnight at 4°C. Negative controls were carried out by omitting the primary antisera or by preabsorbing the specific anti-NOS antibodies with corresponding blocking peptides (Calbiochem) following the protocol indicated by the manufacturer. Immune reaction was revealed by fluorescein isothiocyanate-labeled goat anti-rabbit antibodies (Sigma; working dilution, 1:40) and viewed and photographed under a Zeiss Axioskop fluorescence microscope with a 40x objective (Zeiss, Oberkochen, Germany).

Evaluation of NOS Expression by Western Blot Analysis

Ileal fragments from each mouse were quickly minced and homogenized with a tissue homogenizer (Ing. Terzano, Milan, Italy) in cold lysis buffer with the following composition: 20 mM Tris/HCl pH 7.4, 10 mM NaCl, 1.5 mM MgCl2, 5 mM EGTA, 2 mM Na2EDTA, 1 mM dithiotreitol (DTT), 1 mM PMSF, 1% Triton X-100, 20 µg/ml leupeptin, 1 µg/ml pepstatin, 500 µg/ml pefabloc, and 2.5 µg/ml aprotinin. Upon centrifugation at 17 000 x g at 4°C, the supernatants were collected and the total protein content was measured spectrophotometrically using Bradford reagent (Sigma). The samples, each containing 80 µg of proteins, were electrophoresed by SDS-PAGE (200 V, 1 h) using a denaturating 7.6% polyacrylamide gel with proper molecular weight markers (Bio-Rad, Hercules, CA) and blotted onto nitrocellulose membranes (Amersham Pharmacia Biotech Italy, Cologno Monzese, Italy; 150 V, 1 h). After thorough washings in PBS with 0.1% Tween (T-PBS), the membranes were treated with 5% albumin in T-PBS and incubated overnight at 4°C while being stirred with rabbit polyclonal antibodies against NOS III (Alexis, Läufelingen, Switzerland), NOS II (Alexis), or NOS I (Calbiochem), diluted 1:50 000 in T-PBS with 1% BSA. In our experience, the rabbit polyclonal antibodies against NOS II and III from Alexis yielded more intense and specific staining of the bands than the corresponding antibodies from Calbiochem that were used for immunohistochemistry. The membranes were also immunostained with antiactin antibodies (Zymed, San Francisco, CA; diluted 1:20 000), assuming actin as the control housekeeping protein. Immune reaction was revealed by peroxidase-labeled goat anti-rabbit antibodies (Vector Laboratories, Burlingame, CA) diluted 1:10 000 in T-PBS with 1% BSA and applied to the membranes for 1 h at room temperature while being stirred, followed by a 1-min incubation with the chemiluminescent substrate ECL (Amersham) and exposure to high-sensitivity photographic film (Biomax ML, Kodak, Rochester, NY). For each NOS isoform, quantitative evaluation of the bands appearing on the photographic film was performed by computer-assisted densitometry. The bands, each corresponding to an individual mouse, were digitized with a flatbed scanner, and their optical density was measured using the Scion Image Beta 4.0.2 image analysis program (Scion Corp., Frederick, MD).

Calculations and Statistical Analysis

The quantitative data are given as the mean ± SEM of the values of each experimental group. For functional assays, the values of the treated preparations were expressed as percent change of the basal (or the control) values.

Statistical comparison between the values obtained in control and in relaxin-treated mice was carried out using a two-tailed Student t-test for unpaired values. P < 0.05 was considered significant. The number of the ileal preparations used in each experiment is indicated by n in the results.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ileal Contractile Activity

At basal tension, the ileal preparations from the control mice (n = 12) showed spontaneous and rhythmic phasic contractions (mean amplitude 0.57 ± 0.06 g). This mechanical activity was not influenced by the muscarinic receptor blocker atropine, the neural blocker TTX, or the N-type channel blocker {omega}-conotoxin GVIA (Fig. 1), thus indicating that they are myogenic and not nerve-mediated. Addition of relaxin to the bath medium (n = 8) caused a clear-cut decay of muscle tension (65.1% ± 3.3%) and a reduction in amplitude (35.1% ± 2%) of the spontaneous contractions starting 30–40 min after hormone addition (Figs. 2 and 3). The relaxin-induced depression of ileal motility was long lasting, because it could still be observed 1 h after addition of the peptide to the organ baths, with no spontaneous wearing-off observed by this time (longer times were not observed). The effects of relaxin were not influenced by addition of the neural blocker TTX or the N-type channel blocker {omega}-conotoxin GVIA (data not shown).



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  		FIG. 1. Representative recordings of mechanical activity of ileal mouse preparations 30 min after the noted drugs were added (right panels), none of which are able to cause changes in either basal tension or spontaneous rhythmic phasic contractions



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  		FIG. 2. Representative recordings of mechanical activity of an ileal mouse preparation in A) basal conditions, showing spontaneous phasic contractions; B) 30 min after addition of relaxin, showing a decay of the resting tension and a marked reduction of the contraction amplitude; and C) 10 min after further addition of the NO synthase inhibitor L-NNA, showing an almost complete reversion of the effects of relaxin

Addition of the NOS inhibitor L-NNA to ileal preparations from control mice (n = 4) had no significant effect on either resting tension or amplitude of spontaneous phasic contractions. On the contrary, addition of L-NNA 30 min after relaxin (n = 8) brought both the tissue tension and the amplitude of spontaneous contractions back to basal levels (Figs. 2 and 3). Moreover, relaxin added after a 30-min incubation with L-NNA (n = 4) was unable to influence either the basal tension level or the amplitude of spontaneous contractions (data not shown).

In the ileal preparations from the mice treated long-term with relaxin (n = 4), the amplitude of the spontaneous contractions was markedly reduced (79% ± 3.2%) compared with the control mice (Fig. 4). Addition of L-NNA to the organ bath had no significant effect on the amplitude of the spontaneous contractions, which is at variance with the preparations treated acutely with relaxin (data not shown).



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  		FIG. 4. Representative recordings of spontaneous mechanical activity of ileal preparations from A) control mice and B) mice treated long-term (18 h) with relaxin. Treatment with relaxin causes a marked reduction of ileal contractile activity

In the preparations from the mice treated long-term with relaxin, a further addition of relaxin to the bath medium (n = 4) caused a decay of muscular tension (54.3% ± 2.2%) and a reduction in amplitude of the rhythmic contractions (66.7% ± 3.1%), which was even more prominent than in the acute experiments, up to a near-complete disappearance of spontaneous motility (Fig. 5). Successive addition of L-NNA caused a transient, partial increase of the basal tension and a recovery of the amplitude of the contractions (Fig. 5).



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  		FIG. 5. Mean amplitude of spontaneous contractions of ileal preparations from mice treated long-term with relaxin in A) basal conditions, B) upon acute stimulation with relaxin added to the bath medium, and C) upon further addition of the NOS inhibitor L-NNA. Significance of differences, B vs. C and A, P < 0.05

Ileal Expression of NOS Isoforms

By immunohistochemistry (Fig. 6), both NOS III and NOS II immunoreactivities were expressed by the circular and longitudinal smooth muscle layers of the ileum in control mice. In relaxin-treated mice, the intensity of immunostaining for NOS III and NOS II by smooth muscle cells of the ileal wall appeared clearly increased compared with controls. Scattered NOS I-immunoreactive neurons of the intramural plexuses could be observed in untreated and relaxin-treated mice. There were no apparent differences in the staining intensity of NOS I-positive neurons between controls and relaxin-treated mice.



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  		FIG. 6. Fluorescence micrographs of the ileal wall from control mice (left panel) and relaxin-treated mice (right panel), immunolabeled to reveal endothelial-type NOS III (A and B), inducible NOS II (C and D), and neuronal-type NOS I (E and F). Both NOS III and NOS II are expressed by smooth muscle cells of the longitudinal and the circular layers; the staining intensity is higher in relaxin-treated mice than in controls. NOS I is predominantly expressed by intramural neurons; there are no apparent differences between controls and relaxin-treated mice. Negative controls for the immune reaction as they appear by omitting any primary antiserum in a section from a control mouse (G) or by preabsorbing the anti-NOS III primary antibody with the corresponding blocking peptide in a section from a relaxin-treated mouse (H). Similar images were observed by immunostaining the sections with preabsorbed anti-NOS II and anti-NOS I antisera. Magnifications, A to D, x380; bars = 20 µm; E and F, x950, bars = 10 µm; G and H, x380, bars = 20 µm

By Western blot analysis (Fig. 7), the amount of NOS III and NOS II expressed by the ileum of relaxin-treated mice was significantly higher than in control mice. Conversely, there were no significant differences in the amount of NOS I between the 2 experimental groups.



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  		FIG. 7. Western blot analysis of the expression of NOS isoforms by the ileum of control and relaxin-treated mice. Upper panel shows representative bands from a typical experiment. Expression of actin as control housekeeping protein appears unchanged upon relaxin treatment. Lower panel shows a bar chart of a densitometric analysis of the bands. Relaxin significantly increases the ileal expression of NOS III and NOS II, but not NOS I (open bars, control mice; striped bars, relaxin-treated mice; each bar, n = 6)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study shows that the reproductive hormone relaxin has marked inhibitory effects on mouse small bowel motility, which appear to involve an activation of the intrinsic NO biosynthetic pathway in the smooth muscle coat.

The ileum of mice in proestrous or estrous (i.e., the estrogen-dominated phases of the ovarian cycle) shows a spontaneous motor activity consisting of continuous and rhythmic phasic contractions. These contractions appear to be myogenic and not nerve-mediated because they were not influenced by atropine, TTX, or {omega}-conotoxin GVIA. The motor activity of the isolated ileum was greatly blunted by the addition of relaxin to the bath medium, with a significant decay of basal tension and a reduction of the contraction amplitude fully evident after 30–40 min, and was long-lasting. The neural blockers TTX and {omega}-conotoxin GVIA did not influence the effects of relaxin, thus suggesting that relaxin acts directly on smooth muscle cells, as also observed previously in the myometrium [24]. Rather, the inhibitory action of relaxin on ileal motility is significantly reduced or even abrogated by the NOS inhibitor L-NNA, added either after or before relaxin. These findings indicate that the L-arginine NO pathway is involved in the mechanism of action of relaxin on ileal smooth muscle cells, similar to what occurs in vascular and myometrial smooth muscle cells, which have been also found to be targets for relaxin [23, 24]. The current findings also provide further evidence that relaxin acts as a stimulator of intrinsic NO biosynthesis on its targets, which includes other, different types of cells and organs [40–46] besides smooth muscle cells.

The inhibitory effect of relaxin on ileal motility has been also confirmed by the experiments with mice given relaxin long-term (18 h), in which a marked reduction of the amplitude of spontaneous motor activity was observed as compared with the untreated controls. Further addition of relaxin to the bath medium nearly abolished spontaneous smooth muscle contractions, an effect which could be reverted by the NOS inhibitor L-NNA. Surprisingly, L-NNA was nearly ineffective in modifying the reduction of basal contractile activity, a finding that currently lacks a valid explanation. It has been reported that aortic smooth muscle cells from rats and rabbits can store preformed NO, which can be mobilized under appropriate stimuli to sustain cell relaxation even in the absence of further NO production [47, 48]. Similarly, it is possible that a stored NO pool could also be generated in ileal smooth muscle cells after mice were treated for 18 h with relaxin, thus yielding cell relaxation irrespective of NOS inhibition by L-NNA. Nonetheless, it cannot be excluded that relaxin in the long term may activate additional pathways intrinsic to smooth muscle cells, which cooperate with NO in reducing ileal motility, and which are insensitive to NOS inhibition, as also observed previously in the mouse uterus [24].

In the present study, Western blot analysis indicates that long-term treatment of mice with relaxin causes an overexpression of NOS II and III by the ileum, and immunocytochemistry shows that this phenomenon occurs in the smooth muscle coat. These findings further support the view that relaxin acts on ileal motility by up-regulating the NO biosynthetic pathway and fit well with our previous observations that relaxin increases NOS III immunoreactivity in the myometrium of mice under a similar hormonal regimen [24]. Although it is generally assumed that gastrointestinal smooth muscle cells are a target for NO, whether NOS isozymes are actually expressed by these cells has long been matter of debate, and most previous studies are controversial. Some authors claim that NOS isoforms are not expressed by intestinal smooth muscle cells [49], whereas others, using in situ reverse transcription-polymerase chain reaction, did find NOS III transcripts in these cells [50]. Moreover, functional data exist to indicate that gastrointestinal smooth muscle cells are able to generate NO by a Ca2+-dependent constitutive NOS [51, 52]. In turn, myogenic NO is believed to be involved in the regulation of smooth muscle contractility [53, 54], apparently by acting through the opening of multiple K+ channels on smooth muscle cells and thereby regulating intracellular Ca+2 levels [55]. The present findings provide additional evidence for the expression of NOS II and III by ileal smooth muscle cells and for the physiologic role of myogenic NO in the regulation of intestinal motility. Our findings also suggest that relaxin could be involved in the increased expression of NOS previously observed by other authors in gastrointestinal tissues of pregnant guinea pigs, an effect that had been mainly attributed to the action of estrogens [31, 32].

The present findings may have important physiologic and clinical implications. Previous studies in rats have shown that a reduction of gastric and colonic motility takes place during pregnancy due to increased NO release from the nitrergic component of the nonadrenergic-noncholinergic nerves, and that this mechanism is not operating in the ileum [30]. This finding in the pregnant rat ileum is in keeping with the present observations in mice, in which the inhibitory action of relaxin on ileal motility appears to be exerted directly on smooth muscle cells, and is not nerve-mediated.

Caution is needed when transferring the results obtained in experimental animals to humans. However, inhibition by relaxin of ileal smooth muscle contractility may indeed contribute to an explanation of the reduction of gastrointestinal motility observed in pregnant women and its prompt return to normal values after parturition [4–6], a phenomenon that is paralleled by the trend of circulating relaxin, which attains persistently high levels throughout pregnancy and quickly drops in the postpartum period [18, 19]. It could be speculated that the relaxin-induced decrease of small bowel motility may play a physiologic role during pregnancy, possibly directed to increase the intestinal transit time, thus prolonging the residence time of ingesta in the gut, and consequently increasing the opportunity for absorption of nutrients to fulfill the fuel needs of the mother and the growing fetus. Going a step further, it is possible that an impairment of the physiologic balance between the reproductive hormones acting on bowel motility, including relaxin, may explain the predominant manifestation of functional bowel diseases in women [15] and the cyclic recurrence of gastrointestinal symptoms in women concurrently with the luteal phase of the ovarian cycle [16].



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  		FIG. 3. Mean amplitude of spontaneous contractions of the ileal mouse preparations in A) basal conditions, B) upon addition of relaxin to the bath medium, and C) upon further addition of the NOS inhibitor L-NNA. Significance of differences, B vs. C and A, P < 0.05


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


   FOOTNOTES
 
First decision: 11 July 2001.

1 This study was supported by funds from the University of Florence, Florence, Italy. Back

2 Correspondence: Daniele Bani, Dipartimento di Anatomia, Istologia e Medicina Legale, Sezione di Istologia, V.le G.Pieraccini, 6. I-50139 Firenze, Italy. FAX: 39 055 4271385; bani{at}unifi.it Back

Accepted: October 25, 2001.

Received: June 5, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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