Franco Calamai

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

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 ...

Impaired nitrergic relaxations in the gastric fundus of dystrophic (mdx) mice

Relaxant responses to electrical field stimulation (EFS) were investigated in the gastric longitudinal fundus strips from young normal and mdx dystrophic mice, an animal model of Duchenne muscular dystrophy. In carbachol (CCh) precontracted strips from normal mice, EFS elicited brisk relaxant responses that, depending on stimulation frequency, could be followed by a sustained relaxation. In strips from mdx mice the brisk relaxation was impaired. Smooth muscle responses to direct stimulating agents did not differ in amplitude between the two groups of animals. In strips from both normal and mdx mice, N(G)-nitro-L-arginine (L-NNA) abolished the brisk phase of relaxation, without affecting the sustained response. alpha-chymotrypsin abolished, in both preparations, the sustained relaxant response to EFS as well as relaxation to vasoactive intestinal polypeptide. Results suggest that, in strips from mdx mice, a defective production/release of the neurotransmitter responsible for the brisk relaxation, likely nitric oxide, occurs.

Modulation of nitrergic relaxant responses by peptides in the mouse gastric fundus.

The effects of pituitary adenylate cyclase-activating peptide (PACAP-38) and vasoactive intestinal polypeptide (VIP) were investigated in the gastric fundus strips of the mouse. In carbachol (CCh) precontracted strips, in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-N(G)-nitro arginine (L-NNA) or by the guanylate cyclase inhibitor (ODQ), the sustained one by alpha-chymotrypsin. alpha-Chymotrypsin also increased the amplitude of the EFS-induced fast relaxation. PACAP-38 and VIP caused tetrodotoxin-insensitive sustained relaxant responses that were both abolished by alpha-chymotrypsin. Apamin did not influence relaxant responses to EFS nor relaxation to both peptides. PACAP 6-38 abolished EFS-induced sustained relaxations, increased the amplitude of the fast ones and antagonized the smooth muscle relaxation to both PACAP-38 and VIP. VIP 10-28 and [D-p-Cl-Phe6,Leu17]-VIP did not influence the amplitude of both the fast or the sustained response to EFS nor influenced the relaxation to VIP and PACAP-38. The results indicate that in strips from mouse gastric fundus peptides, other than being responsible for EFS-induced sustained relaxation, also exerts a modulatory action on the release of the neurotransmitter responsible for the fast relaxant response, that appears to be NO.

Depression by Relaxin of Neurally Induced Contractile Responses in the Mouse Gastric Fundus

Abstract The peptide hormone relaxin, which attains high circulating levels during pregnancy, has been shown to depress small-bowel motility through a nitric oxide (NO)-mediated mechanism. In the present study we investigated whether relaxin also influences gastric contractile 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. Evaluation of the expression of nitric oxide synthase (NOS) isoforms was performed by immunohistochemistry and Western blot. In control mice, neurally induced contractile responses elicited by electrical field stimulation (EFS) were reduced in amplitude by addition of relaxin to the organ bath medium. In the presence of the NO synthesis inhibitor l-NNA, relaxin was ineffective. Direct smooth muscle contractile responses were not influenced by relaxin or l-NNA. In strips from relaxin-pretreated mice, the amplitude of neurally induced contractile responses was also reduced in respect to the controls, while that of direct smooth muscle contractions was not. Further addition of relaxin to the bath medium did not influence EFS-induced responses, whereas l-NNA did. An increased expression of NOS I and NOS III was observed in gastric tissues from relaxin-pretreated mice. In conclusion, the peptide hormone relaxin depresses cholinergic contractile responses in the mouse gastric fundus by up-regulating NO biosynthesis at the neural level.

Gastric motor responses elicited by vagal stimulation and purine compounds in the atropine‐treated rabbit

1 The effects of vagal inhibitory stimulation and of purine compounds were studied in the rabbit stomach. 2 Gastric motility was assessed by the balloon method. Vagal nerves were electrically stimulated at the neck. Purine compounds were injected intra‐arterially. 3 In the atropine‐treated rabbit, vagal stimulation caused relaxant motor responses followed by a rebound contraction. 4 Among the purine compounds, only ADP and ATP caused relaxant motor responses similar to the effects of vagal inhibitory stimulation. However, the relaxation produced by ATP was more powerful than that due to ADP, especially at lower infusion rates. 5 Vagal inhibitory responses were recorded during and after infusion of ATP. When relaxation by ATP was fully developed, vagal inhibitory stimulation was ineffective. At the highest infusion rates of ATP, a depression of the vagal inhibitory motility was also observed after cessation of the infusion. 6 Relaxant responses to ATP and vagal inhibitory stimulation were not influenced by theophylline, scarcely affected by α,β‐methylene ATP, but were reduced or blocked by reactive blue 2. 7 The results are consistent with ATP being an inhibitory neurotransmitter in the stomach of the rabbit.

Influence of relaxin on the neurally induced relaxant responses of the mouse gastric fundus

Abstract 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.

The influence of the vagally induced rebound contractions on the non-adrenergic, non-cholinergic (NANC) inhibitory motility of the rabbit stomach and the role of prostaglandins

Factors influencing the vagally induced rebound contraction and its role in gastric inhibitory motility were studied in the anaesthetised rabbit. Gastric motility was assessed from measurements of gastric volume by means of an intragastric balloon. In the atropine- and guanethidine-treated animals, vagal stimulation caused biphasic motor responses: a rapid relaxation was followed by a rebound contraction. The latter, depending on experimental conditions, was able to restore and maintain gastric volume at the basal level. However, the rebound contraction was greatly influenced by the stimulation parameters and the basal gastric volume. Stimulation periods of less than 30 sec, or stimulation frequencies above 20 Hz, as well as basal gastric volumes above 70 ml could reduce the amplitude of the post-stimulus excitatory motility, and transformed the biphasic response into a triphasic one: a slow, long-lasting relaxation appeared after the rebound contraction. Prostaglandin-synthesis inhibitors of the non-steroidal anti-inflammatory group depressed the rebound contraction, and caused persistence of gastric relaxation, even after the offset of vagal stimulation. PGE2 evoked excitatory motor responses which closely mimicked the vagally induced rebound contraction. PGE2 also interrupted the rapid or slow, long-lasting relaxant responses. PGF2 alpha elicited tonic excitatory responses. These results suggest that PGE2 is involved in the mechanism underlying post-stimulus excitatory motility. They also suggest that the rebound contraction is a key factor in determining the inhibitory motility pattern of the rabbit stomach.

Influence of orexin A on the mechanical activity of mouse gastric strips

The presence of orexins and orexin receptors has been revealed not only in the central nervous system but also in the gastrointestinal tract. The present study was aimed to investigate the influence of orexin A (OXA) on the mechanical activity of fundal and antral strips of the mouse stomach. In the fundus, electrical field stimulation (EFS) elicited tetrodotoxin (TTX)-sensitive, frequency-dependent contractile responses whose amplitude was markedly reduced by OXA and enhanced by the orexin-1 type receptor antagonist SB-334867. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), OXA was no longer effective. Methacholine caused a sustained contracture whose amplitude was not influenced by OXA, TTX or L-NNA. In carbachol-precontracted strips, the neurally-induced relaxant responses elicited during EFS were increased in amplitude by OXA. Antral strips showed a spontaneous contractile activity that was unaffected by TTX or L-NNA and transiently depressed by EFS. OXA did not influence either the spontaneous motility or the EFS-induced effects. The results indicate that OXA exerts region-specific effects and that, in the fundus, depresses EFS-induced contractile responses by acting at the nervous level. It is likely that NO is involved in the effects of the peptide.

Relaxin Restores Altered Ileal Spontaneous Contractions in Dystrophic (mdx) Mice

Abstract: We studied the effects of relaxin on ileal contractility in normal and dystrophic (mdx) mice. Ileal preparations from male normal and mdx mice showed spontaneous myogenic contractions whose amplitude was significantly higher in the latter ones. Relaxin added to the bath medium together with l‐arginine depressed the amplitude of the spontaneous contractions in the mdx mice to a level similar to that of the normal mice. The nitric oxide synthase (NOS) inhibitor L‐NG‐nitroarginine reverted this effect. In mdx mice pretreated for 18 hours with relaxin, spontaneous motility was greatly reduced in amplitude, resembling that of the normal mice. Concurrently, iNOS expression in the muscle coat was markedly increased. Therefore, in mdx mice, relaxin can restore an ileal motility pattern similar to that of the normal mice by upregulating endogenous NO biosynthesis.

Gastric relaxation in response to chemical stimulation of the area postrema in the rabbit

Microinjections of DL-homocysteic acid into the area postrema (AP) of anesthetized rabbits provoked gastric relaxations associated with small changes in blood pressure and marked excitatory effects on respiration. Both gastric and cardiovascular effects failed to occur after bilateral vagotomy. Comparable gastric relaxations were induced before and after treatment with atropine or atropine and guanethidine. The AP appears to play a role in gastric motility via vagus nerves and nonadrenergic noncholinergic intramural inhibitory neurons.