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BOR - Papers in Press, published online ahead of print June 23, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029579
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BIOLOGY OF REPRODUCTION 71, 1325–1329 (2004)
DOI: 10.1095/biolreprod.104.029579
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Influence of Relaxin on the Neurally Induced Relaxant Responses of the Mouse Gastric Fundus1

Maria Caterina Baccari2,3, Daniele Bani4, Mario Bigazzi5, and Franco Calamai3

Departments of Physiological Sciences3 and Anatomy, Histology and Forensic Medicine,4 University of Florence, 50134 Florence, Italy Prosperius Institute,5 50125 Florence, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The peptide hormone relaxin has been reported to depress the amplitude of contractile responses in the mouse gastric fundus by upregulating nitric oxide (NO) biosynthesis at the neural level. In the present study, we investigated whether relaxin also influenced nonadrenergic, noncholinergic (NANC) gastric relaxant responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 µg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. In carbachol precontracted strips from control mice and in the presence of guanethidine, electrical field stimulation (EFS) elicited fast relaxant responses that may be followed by a sustained relaxation. All relaxant responses were abolished by tetrodotoxin. Relaxin increased the amplitude of the EFS-induced fast relaxation without affecting either the sustained one or the direct smooth muscle response to papaverine. In the presence of the NO synthesis inhibitor L-NG-nitro arginine (L-NNA), that abolished the EFS-induced fast relaxation without influencing the sustained one, relaxin was ineffective. In strips from relaxin-pretreated mice, EFS-induced fast relaxations were enhanced in amplitude with respect to the controls, while sustained ones as well as direct smooth muscle responses to papaverine were not changed. Further addition of relaxin to the bath medium did not influence neurally induced fast relaxant responses, whereas L-NNA did. In conclusion, in the mouse gastric fundus, relaxin enhances the neurally induced nitrergic relaxant responses acting at the neural level.

mechanisms of hormone action, nitric oxide, relaxin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clinical evidence and experimental studies indicate that female sex hormones can affect gut motility [1] and increased levels of estrogens and/or progesterone have been suggested to be involved in gastrointestinal motor dysfunctions [2–4] frequently observed in the pregnant state. Even if the issue of which female hormones are involved in these dysmotilities is still a matter of debate, recent studies suggest that estrogens can modulate nitric oxide (NO) production: under the influence of estrogens, an increased NO release from enteric nerves could be responsible for or contribute to the delayed gastric emptying and increased colonic transit time frequently observed in pregnancy [5]. In fact, NO released from nonadrenergic, noncholinergic (NANC) nerves is considered the major inhibitory neurotransmitter that depresses cholinergic contractions and causes relaxation of the gut [6–10]. In pregnancy, an increased NOS expression and NO production have been demonstrated in several organs and structures [5, 11, 12].

Besides sex steroids hormones, evidence is accumulating that the peptide hormone relaxin is also able to stimulate NO biosynthesis in a variety of tissues [13–16]. In particular, in the gastrointestinal tract, relaxin has been shown to markedly decrease both spontaneous contractions in the ileum [17] and neurally induced cholinergic contractions in the stomach [18].

However, no data exist on the effects of this hormone on the relaxant responses of the gut. Because it has been recently reported that, in pregnant animals, gastric relaxations are higher in amplitude than in the nonpregnant [19], the aim of this work is to verify if relaxin, which attains high levels in pregnancy [20, 21], can influence the relaxant responses in the mouse stomach.


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

Virgin albino female mice of Swiss strain, 8–12 wk old and weighing about 30 g (Morini, Reggio Emilia, Italy), were employed. The mice were fed standard laboratory chow and water, and were housed under a 12L: 12D photoperiod and controlled temperature (21 ± 1°C). 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). After a 1-wk acclimatization, the mice underwent assessment of the phase of the estrous cycle by light microscopic examination of vaginal smears stained with Papanicolaou, according to Austin and Rowlands [22]. Only mice in proestrus or estrus, that is, the estrogen-dominated phases, entered the experiments. The reason for this choice is that, in several relaxin target organs and tissues, estrogens are required to induce relaxin responsiveness [23, 24]. The mice were randomly distributed in two groups, 12 animals each. The mice of the first group received a single subcutaneous injection of 1 µg of highly purified porcine relaxin (2500–3000 U/mg), prepared according to Sherwood and O'Byrne [25]. As previously reported [14, 17, 18], the hormone was dissolved in 0.2 ml of benzopurpurin (Fluka AG, Buchs, Switzerland) (1% w/v) in phosphate-buffered saline (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 to those used in previous studies in mice and proved effective in inducing clear-cut responses in target organs, in terms of cell growth [24] or NOS induction [14, 17, 18]. The mice of the second group received the vehicle alone. They are referred to as control mice. Eighteen hours later, the mice were killed by cervical dislocation and the stomach was rapidly dissected free from the abdomen. An 18-h exposure to relaxin of mice in proestrus or estrus was deemed adequate to obtain a prolonged stimulation [17, 18]. Additional experiments were carried out in three relaxin pretreated mice in proestrus to determine the plasma levels of the hormone: circulating relaxin in blood samples taken 18 h from the injection reached the value of 152.1 ± 42.2 pg/ml, as evaluated by ELISA using a commercially available kit (Immundiagnostik, Bensheim, Germany). Taking into account the duration of the different phases of the estrous cycle in mice [26], this exposure time also allows the animals not to enter diestrus, the progesterone-dominated phase.

Functional Studies

The stomach was placed on a Petri dish containing warm (37°C) Krebs-Henseleit solution of the following composition (mM): NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, CaCl2 2.5, and glucose 10, gassed with 95% O2:5% CO2 (pH 7.4). Two full-thickness tissue strips (2 mm x 10 mm) were cut from each fundus region in the direction of the longitudinal muscle. One end of each strip was tied to a platinum rod while the other was connected to a force displacement transducer (Grass model FT03, Quincy, MA) by a silk thread for continuous recording of isometric tension. The transducer was coupled to a polygraph (Sanborn model 7700, Waltham, MA). The fundal strips were mounted in 5-ml double-jacketed organ baths containing Krebs-Henseleit solution gassed with a 95% O2:5% CO2 mixture. Prewarmed water (37°C) was circulated through the outer jacket of the tissue bath via a constant-temperature circulator pump. The temperature of the Krebs-Henseleit solution in the organ bath was maintained within ±0.5°C. As previously reported [18, 27, 28], tissues were allowed to equilibrate for 1 h under an initial load of 0.8 g. During this period, repeated and prolonged washes of the preparations with Krebs-Henseleit solution were done to avoid accumulation of metabolites in the organ baths.

Electrical field stimulation (EFS) was applied via two platinum wire rings (2-mm diameter, 5 mm apart) through which the fundal strip was threaded. Electrical impulses (rectangular waves, 80 V, 4–16 Hz, 0.5 msec, for 15 sec) were provided by a Grass model S8 stimulator.

Drugs

The following drugs were used: the cholinergic agonists carbamylcholine chloride (carbachol, CCh 1 x 10–6 M), the adrenergic blocker guanethidine monosulfate (1 x 10–6 M), the neural blocker tetrodotoxin (TTX, 1 x 10–6 M), the NOS inhibitor NG-nitro-L-arginine (L-NNA, 2 x 10–4 M), relaxin (3 x 10–8 M), the smooth muscle relaxant agent papaverine (1 x 10–5 M). All drugs were obtained from Sigma Chemical Co. (St. Louis, MO), except for relaxin (pure porcine relaxin, 2500–3000 U/ mg) that was generously provided by Dr. O.D. Sherwood, University of Illinois (Urbana, IL). Solutions were prepared on the day of the experiment, except for TTX, for which a stock solution was kept stored at –20°C. Drug concentrations are given as final bath concentrations. The concentrations of L-NNA and relaxin used were in the range of those previously shown to be effective in mice to inhibit NO-mediated gastric relaxation [27, 28] and gastric, ileal, and myometrial contractile activity [14, 17, 18], respectively. Drugs were dissolved in Krebs-Henseleit solution and were added in volumes not exceeding 0.5% of the bath volume.

In a first series of experiments, the effects of EFS were studied in strips from control mice. To establish the neurally induced NANC relaxant responses, EFS was applied in the presence of guanethidine and CCh, to rule out the adrenergic and the cholinergic influence, respectively. EFS was applied when contraction following CCh administration reached a stable plateau phase. The interval between two subsequent applications of CCh was no less than 15 min, during which repeated and prolonged washes of the preparations with Krebs-Henseleit solution were performed. To test the neural component of the relaxant responses, in some strips, tetrodotoxin was applied to the bath medium 10 min before performing EFS. The influence of relaxin or L-NNA on neurally induced relaxant responses to EFS (4–16 Hz) were first investigated separately on different strips, starting 10 min after reagent addition to the bath medium, then in combination to study the reciprocal influence of the two substances on the relaxant responses. In these latter experiments, L-NNA was added to the bath medium 20 min following relaxin or, in a different set of strips, relaxin was added to the bath medium 10–15 min following L-NNA.

The effects of relaxin were also investigated on direct smooth muscle relaxant responses that were elicited in CCh precontracted strips by the addition of the relaxant agent papaverine (1 x 10–5 M) to the bath medium.

The previously described series of experiments were also carried out on fundal strips from relaxin-pretreated mice.

Data Analysis and Statistical Test

Relaxant responses are expressed as percent decrease of the muscular tension induced by 1 x 10–6 M CCh. Amplitude values of fast relaxations following EFS refer to the maximal peak obtained during the stimulation period. Amplitude values of slow relaxations following EFS refer to the maximal peak obtained following the stimulation period as compared with the prestimulus level.

All values are expressed as mean ± SEM. The number of preparations is designated by n in the results.

Statistical analysis was performed by means of Student t-test to compare two experimental groups or of one-way ANOVA when more than two groups were compared. When ANOVA indicated that significant differences existed, multiple comparisons between groups was carried out by Newman-Keuls posttest. For all statistical tests, values were considered significantly different with P values equal or less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Relaxant Responses of Strips from Control Mice

At basal tension, addition of CCh (1 x 10–6 M) to the bath medium (n = 20) caused a rapidly arising contraction (mean amplitude 1.29 ± 0.10 g), which persisted until washout. In CCh (1 x 10–6 M) precontracted strips and in the presence of guanethidine (1 x 10–6 M), EFS elicited relaxant responses the amplitude of which increased by increasing the stimulation frequency (Fig. 1), as previously observed [27, 28]. At the lowest stimulation frequency employed (4 Hz), the inhibitory responses consisted of a fast relaxation followed by a rapid return of strip tension to baseline at the end of the stimulation period. By increasing the stimulation frequency, strip tension did not immediately return to the baseline at the end of the stimulation period but a lower-amplitude, sustained relaxation was observed (Fig. 1). TTX (1 x 10–6 M) abolished all the relaxant responses to EFS (P < 0.05; data not shown), thus indicating that they were neurally induced.



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  		FIG. 1. Relaxant responses elicited by EFS in CCh (1 x 10–6 M)-precontracted gastric fundal strips from control mice. A) Typical tracings showing relaxant responses to EFS. Compared with the control responses (left record), addition of relaxin (3 x 10–8 M, center record) potentiates the amplitude of the EFS-induced fast relaxant responses in the whole range of stimulation frequency employed. The NO synthesis inhibitor L-NNA (2 x 10–4 M), added to the bath medium 20 min after relaxin (right record), causes the abolition of the neurally induced fast relaxations without affecting the sustained ones. The three records are obtained from the same strip. B) Bar chart of the mean amplitude of EFS-induced fast relaxant responses at different stimulation frequencies. Amplitude results refer to the maximal peak with respect to prestimulus level and represent percent decreases relative to the tension induced by CCh (1 x 10–6 M). Relaxin (3 x 10–8 M), 20 min after its addition to the bath medium, increases the mean amplitude of the fast relaxant responses in the whole range of stimulation frequency employed. Subsequent addition of L-NNA (2 x 10–4 M) to the bath medium in the presence of relaxin causes, after 10 min, the abolition of the EFS-induced fast relaxant responses. All values are means ± SEM of six preparations. *, P < 0.05 versus the controls (one-way ANOVA and Newman-Keuls posttest)

Addition of relaxin (3 x 10–8 M) to the bath medium (n = 10) did not influence strip tension but caused a marked increase in amplitude of the EFS-induced fast inhibitory responses (Fig. 1) in 8 out of the 10 strips examined, without affecting sustained inhibitory responses (Table 1). The effects of relaxin on fast inhibitory responses were observed over the whole range of stimulation frequency employed (Fig. 1). The effects of relaxin, already appreciable 10 min after its addition to the bath medium, were fully evident after 25–30 min.


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  		TABLE 1. Mean amplitude and duration of EFS-induced sustained relaxations.a

Addition of the NOS inhibitor L-NNA (2 x 10–4 M) to the bath medium (n = 8) caused the abolition of the fast inhibitory responses in the whole range of stimulation frequency employed, but did not influence the amplitude of the sustained relaxations (Table 1; Fig. 1A). The effects of L-NNA, which were already present 10 min after its addition to the bath medium, were also observed in the presence of relaxin (3 x 10–8 M) (Fig. 1). Relaxin (3 x 10–8 M) added to the bath medium 10 min after L-NNA (2 x 10–4 M) (n = 4), when the effects of the NOS inhibitor on EFS-induced fast inhibitory responses were manifested, did not revert the effects of L-NNA (P > 0.05) (data not shown).

Direct smooth muscle relaxation was achieved by addition of papaverine (1 x 10–5 M) to the bath medium (n = 6), which caused a slowly developing sustained relaxation (mean amplitude 69.7 ± 2%) that persisted until washout (5 min, longer time not observed). The amplitude of the smooth muscle relaxant responses caused by papaverine was not affected (P > 0.05) by relaxin (3 x 10–8 M) (data not shown), thus indicating that direct smooth muscle responses were not influenced by this hormone.

Relaxant Responses of Strips from Relaxin-Pretreated Mice

At basal tension, addition of CCh (1 x 10–6 M) to the bath medium (n = 20) caused a sudden rise in strip tension, the amplitude (mean amplitude 1.30 ± 0.12 g) of which was not different compared with the control mice (P > 0.05).

In CCh-precontracted strips and in the presence of guanethidine (1 x 10–6 M), EFS induced fast and sustained relaxations (Fig. 2), as occurred in the controls. However, as compared with the controls, (P < 0.05) the fast relaxant responses were greater in amplitude (Fig. 3) in the whole range of stimulation frequency employed (Fig. 2A compare with Fig. 1A), while sustained ones were not different (P > 0.05).



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  		FIG. 2. Relaxant responses elicited by EFS in CCh (1 x 10–6 M)-precontracted gastric fundal strips from relaxin-pretreated mice. A) Typical tracings showing relaxant responses to EFS. Fast relaxant responses (left record) appeared enhanced in amplitude compared with those obtained in strips from control mice (Fig. 1A, left trace). Relaxin (3 x 10–8 M, center record) does not influence the amplitude of the fast relaxant response. The NOS inhibitor L-NNA (2 x 10–4 M), added to the bath medium 20 min after relaxin (right record), causes the abolition of the neurally induced fast relaxations, without affecting the sustained ones. The three records are obtained from the same strip. B) Bar chart of the mean amplitude of EFS-induced fast relaxant responses at different stimulation frequencies. Amplitude results refer to the maximal peak with respect to prestimulus level and represent percent decreases relative to the tension induced by CCh (1 x 10–6 M). Relaxin (3 x 10–8 M), 20 min after its addition to the bath medium, does not influence the mean amplitude of the EFS-induced fast relaxant responses in the whole range of stimulation frequency employed. Subsequent addition of L-NNA (2 x 10–4 M) to the bath medium in the presence of relaxin abolishes, after 10 min, the fast relaxations. All values are means ± SEM of six muscle strip preparations. *, P < 0.05 versus the controls (one-way ANOVA and Newman-Keuls posttest)



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  		FIG. 3. Comparison among the mean amplitude of EFS-induced fast relaxant responses in CCh (1 x 10–6 M)-precontracted gastric fundal strips from control and relaxin-pretreated mice. Bar chart of the mean amplitude of EFS-induced fast relaxant responses at different stimulation frequencies. Amplitude results refer to the maximal peak with respect to prestimulus level and represent percent decreases relative to the tension induced by CCh (1 x 10–6 M). The mean amplitude of the EFS-induced fast relaxant responses is increased, in the whole range of stimulation frequency employed, either in strips from control animal 20 min following the addition of relaxin (3 x 10–8 M) (dark columns) or in strips from relaxin-pretreated mice (dashed line columns) in respect to the controls (open columns). All values are means ± SEM of six preparations. *, P < 0.05 versus the controls; **, P < 0.05 versus both control and addition of relaxin to the bath medium (one-way ANOVA and Newman-Keuls posttest)

Further addition of relaxin (3 x 10–8 M) to the bath medium (n = 8) did not affect the amplitude of either the fast or the sustained relaxant responses to EFS (Fig. 2).

Addition of L-NNA (2 x 10–4 M) to the bath medium (n = 6) abolished the EFS-induced fast relaxant responses (P < 0.05), even in the presence of relaxin (P < 0.05) (Fig. 2).

Addition of papaverine (1 x 10–5 M) to the bath medium (n = 6) caused a slowly developing, sustained relaxation that persisted until washout (5 min, longer time not observed), as in the control strips. Direct smooth muscle responses were not influenced by relaxin pretreatment: the mean amplitude of the papaverine-induced relaxations (67.7 ± 3%) was not different as compared with the control mice (P > 0.05; data not shown), thus indicating that smooth muscle responses were not impaired.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several lines of evidence indicate a close link between the peptide hormone relaxin and NO production in a variety of tissues and organs [13–16], and recent studies have shown that relaxin may influence the contractile responses in the gastrointestinal tract by modulating NO biosynthesis [17, 18]. The present study shows that relaxin can also influence relaxant responses of the stomach by a NO-mediated pathway. In the mammalian stomach, evidence has been given that several types of relaxant responses can be elicited, either in vivo or in vitro, by NANC nerve activation and that several types of inhibitory neurotransmitter are involved in gastric relaxation [29–35].

In the present experiments and in previous studies [27, 28], two components in the NANC-mediated responses were observed in strips from the gastric fundus of mice upon EFS: a fast relaxation during the stimulation period, which can be followed by a sustained relaxant response after the end of the stimulation, depending on the stimulation frequency. The fast relaxation involved NO as an inhibitory neurotransmitter because it was abolished by L-NNA, whereas the sustained relaxation was NO-independent because it was unaffected by this NOS inhibitor. The results of the present study indicate that relaxin influences the fast component of the relaxant responses likely acting on nitrergic nerves. In fact, relaxin enhanced the amplitude of the neurally-induced fast relaxation, but was ineffective on the sustained, NO-independent relaxant responses, as well as on the direct smooth muscle relaxations induced by papaverine. The observation that relaxin was ineffective in the presence of L-NNA further supports the view that the effects of relaxin are mediated through an increased NO biosynthesis and release. The modulatory action exerted by relaxin on the nitrergic component of NANC nerves is in keeping with our previous finding of an overexpression of NOS I and NOS III in the stomach of relaxin-pretreated animals [18] and with the results obtained in the present study in strips from the relaxin-pretreated mice. In fact, in these latter preparations, we observed an increased amplitude of the EFS-induced fast inhibitory responses, whereas sustained ones and direct smooth muscle responses to the relaxant agent papaverine were unchanged as compared with the control animals. The lack of effects of the addition of relaxin to the bath medium in strips from relaxin-pretreated mice may suggest that the effects of the hormone given systemically were already fully manifested and could not be further influenced by the relaxin given in the bath, as shown in Figure 3. This finding confirms our previous observations [18].

Even if the influence of sex hormones and pregnancy on gastric motility and emptying is not definitively established [1, 2, 36–39], the relaxin-induced increase in amplitude of the nitrergic fast relaxations may have some physiological implications. In fact, recent studies have shown that enhanced nitrergic relaxations can be observed in the stomach of pregnant animals and that estrogens could enhance gastric transit time by increasing the amplitude of the relaxant responses [19]. To date, estrogens have been also held responsible for the increased expression of NOS observed in pregnancy [5, 11]. The present results, together with the previous ones [18], indicate that, besides estrogens, relaxin can also contribute to the upregulation of the NO biosynthetic pathway. Therefore, more than one female sex hormone may contribute to the increase of NOS expression and NO production occurring in pregnancy.

The exact role of the nitrergic NANC innervation and of the relaxant responses in gastric transit time is far from being clarified [19, 38, 40, 41]. Nevertheless, it is widely accepted that an altered NO production may lead to gastrointestinal dysmotilities [42]. Therefore, even if caution is mandatory because in vitro studies do not necessarily reflect the in vivo gastric motor functions [41], we can tentatively speculate that an increased adaptive fundic relaxation and a decrease of the contractile activity of the stomach caused by an increased NO release by NANC nerves could be the basis for a delayed gastric emptying, as observed in pregnancy.

The more knowledge of the interactions between the neural and the hormonal control of the gastric motility, the better will be our understanding of the mechanisms underlying gastrointestinal disorders, which might eventually contribute to the development of new therapeutic approaches. In this view, the emerging role played by relaxin as a physiologic modulator of the nitrergic transmission deserves to be further investigated.


   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.


   FOOTNOTES
 
1 Supported by funds from the University of Florence, Florence, Italy. The financial support of Telethon-Italy (grant GGP02152) is also gratefully acknowledged. Back

2 Correspondence: Maria Caterina Baccari, Department of Physiological Sciences, University of Florence, Viale G.B. Morgagni 63, I-50134 Florence, Italy. FAX: +39 055 437 9506; mcaterina.baccari{at}unifi.it Back

Received: 11 March 2004.

First decision: 24 April 2004.

Accepted: 9 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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M. C. Baccari, S. Nistri, M. G. Vannucchi, F. Calamai, and D. Bani
Reversal by relaxin of altered ileal spontaneous contractions in dystrophic (mdx) mice through a nitric oxide-mediated mechanism
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2007; 293(2): R662 - R668.
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