Reversal by relaxin of altered ileal spontaneous contractions in dystrophic (mdx) mice through a nitric oxide-mediated mechanism.

Altered nitric oxide (NO) production/release is involved in gastrointestinal motor disorders occurring in dystrophic (mdx) mice. Since the hormone relaxin (RLX) can upregulate NO biosynthesis, its effects on spontaneous motility and NO synthase (NOS) expression in the ileum of dystrophic (mdx) mice were investigated. Mechanical responses of ileal preparations were recorded in vitro via force-displacement transducers. Evaluation of the expression of NOS isoforms was performed by immunohistochemistry and Western blot. Normal and mdx mice were distributed into three groups: untreated, RLX pretreated, and vehicle pretreated. Ileal preparations from the untreated animals showed spontaneous muscular contractions whose amplitude was significantly higher in mdx than in normal mice. Addition of RLX, alone or together with l-arginine, to the bath medium depressed the amplitude of the contractions in the mdx mice, thus reestablishing a motility pattern typical of the normal mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) or the guanylate cyclase inhibitor ODQ reversed the effects of RLX. In RLX-pretreated mdx mice, the amplitude of spontaneous motility was reduced, thus resembling that of the normal mice, and NOS II expression in the muscle coat was increased in respect to the vehicle-pretreated mdx animals. These results indicate that RLX can reverse the altered ileal motility of mdx mice to a normal pattern, likely by upregulating NOS II expression and NO biosynthesis in the ileal smooth muscle.

Relaxin: new functions for an old peptide.

The peptide relaxin (RLX) was one of the first hormones to be described with a specific function in parturition. In the past ten years, there has been a revaluation of RLX physiology and the concept that sex hormones play roles that are limited to reproductive functions is rapidly changing. In this view, growing evidence indicates that the peptide hormone RLX, structurally related to insulin and insulin-like growth factor and primarily secreted by the corpus luteum during pregnancy, besides well demonstrated actions on reproductive tissues, is involved in a variety of functions. Among them, RLX influences the brain and regulates pituitary hormone secretion, causes renal vasodilatation, increases coronary flow, exerts chronotropic action on the heart and affects gastrointestinal motor responses. Recent studies suggest that in several smooth muscles the hormone appears to act by promoting the biosynthesis of nitric oxide (NO), whose altered production may be involved in smooth muscle dysmotilities. The recent cloning of the RLX receptors and studies on their possible signal transduction mechanisms are stimulating researchers to further investigate the effects of this hormone and its mechanism of action. This may lead to the discovery of agonists and antagonists for RLX and the development of new therapeutic approaches in some human diseases. The aim of this mini-review is to summarize the most recent findings on the multiple actions of RLX hoping to bring a contribution for the future perspectives in this field.

Preliminary results on the prediction of countershock success with fibrillation power.

In a study of artifact-free ventricular fibrillation episodes in 54 patients, 28 of whom experienced return of spontaneous circulation (ROSC), the power of different indicators to predict the ROSC was compared. Taking the average of sensitivity, specificity and positive and negative predictive value, the dominant frequency reaches 76%, the mean amplitude 72% and fibrillation power 71%. There is little correlation between the three indicators.

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.

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.

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 microg) 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.

Nitric oxide as modulator of cholinergic neurotransmission in gastric muscle of rabbits.

The effects of the nitric oxide (NO) synthesis inhibitors, NG-nitro-L-arginine (L-NNA) and NG-nitro-L-arginine methyl ester (L-NAME), on the electrical field stimulation (EFS)-induced inhibitory responses were investigated. EFS caused, in strips contracted by means of substance P (SP), prostaglandin F2 alpha (PGF2 alpha), or carbachol (CCh), a fast relaxant response that, depending on stimulation frequency and strip tension, could be followed by a slower, sustained relaxation. The NO synthesis inhibitors blocked the EFS-induced fast relaxations and often reversed them into contractions; these effects were greatly counteracted in SP- or PGF2 alpha-treated strips by scopolamine or atropine. In CCh-precontracted strips, either L-NNA or L-NAME became progressively unable to block the EFS-induced fast relaxations as the CCh concentration was increased. The NO synthesis inhibitors greatly reduced the sustained relaxant responses elicited either by EFS or exogenous vasoactive intestinal polypeptide (VIP). The results indicate that the NO synthesis inhibitors abolish the neurally induced fast relaxation by interfering with the cholinergic excitatory pathway. The involvement of both VIP and NO in sustained relaxations is also suggested.

Prostaglandin E2 modulates neurally induced nonadrenergic, noncholinergic gastric relaxations in the rabbit in vivo.

BACKGROUND & AIMS: Prostaglandin (PG) E2 has been shown to modulate adrenergic and cholinergic neurotransmission in the gut. This study investigated PGE2 influence on vagally induced, nonadrenergic, noncholinergic (NANC) gastric relaxations. METHODS: Mechanical activity of the stomach was recorded in anesthetized rabbits. RESULTS: In atropine-treated animals, electrical vagal stimulation or arterial bolus injection of the ganglion stimulant dimethyl phenylpiperazinium iodide (DMPP) evoked inhibitory responses that varied from a brisk relaxation, interrupted by a poststimulus excitatory motility (biphasic response), to a long-lasting relaxation (monophasic response). PGE2 reduced and, at the highest doses, abolished the neurally induced relaxant responses elicited either by vagal stimulation or DMPP administration but did not affect the gastric relaxation caused by adenosine triphosphate (ATP), sodium nitroprusside (SNP), or vasoactive intestinal polypeptide (VIP). ATP or 2-methylthioadenosine triphosphate (2-MeSATP) reduced and then suppressed vagally induced inhibitory motility; the relaxant responses elicited by SNP, VIP, and ATP itself were not influenced. After administration of the prostaglandin synthesis inhibitor suprofen, ATP and 2-MeSATP failed to block vagally induced inhibitory responses. Arterial infusion of adenosine at the highest rates did not influence the amplitude of the vagally induced relaxant responses. Following theophylline administration, ATP still blocked the relaxation elicited by vagal stimulation. CONCLUSIONS: PGE2 may modulate NANC inhibitory neurotransmission in the stomach. The effects of ATP on the neurally induced NANC gastric relaxation may be caused by PGE2.

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.

Modulation of cholinergic transmission by nitric oxide, VIP and ATP in the gastric muscle.

The influence of the putative inhibitory neurotransmitters nitric oxide (NO), vasoactive intestinal polypeptide (VIP) and adenosine 5'-triphosphate (ATP) was examined on the contractile responses elicited, either by electrical field stimulation (EFS) or exogenous acetylcholine (Ach), in strips of the circular muscle of the rabbit gastric corpus. Muscular contractions evoked by Ach were not influenced by the NO-releasing compound sodium nitroprusside (SNP), but were depressed by VIP and scarcely affected by ATP. In contrast, the putative inhibitory neurotransmitters all depressed or even blocked the neurally induced cholinergic contractions elicited by EFS. Therefore, NO, VIP or ATP, besides causing muscular relaxation, may modulate the cholinergic transmission at the pre- and/or post-junctional level in the nerve-muscle pathway.

Effects of L-NG-nitro arginine on cholinergic transmission in the gastric muscle of the rabbit.

In the circular muscle of the rabbit gastric corpus, the nitric oxide-synthesis inhibitor L-NG-nitro arginine (L-NOARG), enhanced the neurally-induced cholinergic responses evoked by electrical field stimulation (EFS) and ganglionic stimulating agents (nicotine, dimethylphenyl piperazinium iodide). The muscular contractions caused by acetylcholine (Ach) and methacholine were not influenced by the nitric oxide-synthesis inhibitor. The nitric oxide-releasing compound sodium nitroprusside (SNP) did not affect the Ach-induced muscular responses. Our results suggest that L-NOARG enhances gastric cholinergic responses by removing an inhibitory influence exerted at the prejunctional level in the nerve-muscle pathway.

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.

Vagally-induced non-adrenergic, non-cholinergic inhibitory motility in the rabbit stomach "in vivo".

In atropine-treated rabbits, electrical stimulation of vagal nerves at low frequency and low basal gastric volumes evoked a biphasic response: rapid onset relaxation followed by rebound contractions. Increase in the frequency of stimulation and/or in basal gastric volume caused a depression of the rebound contraction and a conversion of the biphasic response into a triphasic one: i.e., the post-stimulus excitatory motility was followed by a long-lasting slow relaxation. The rebound contraction, which was mimicked by arterial injections of prostaglandin E2, appears to be a key factor in the conversion of one type of response into the other.

Effects of arterial infusions of adenosine 5'-triphosphate (ATP) and vasoactive intestinal polypeptide (VIP) on vagal excitatory motor responses in the rabbit stomach 'in vivo'.

In the rabbit stomach 'in vivo' adenosine and adenosine 5'-triphosphate depressed the contractile activity elicited by vagal stimulation. The order of potency was ATP greater than adenosine. Only ATP completely blocked the excitatory motor responses. Vasoactive intestinal polypeptide injected close-arterially scarcely affected the vagal contractile activity of the stomach.

Non-adrenergic, non-cholinergic influences on rabbit gastric tone.

In the rabbit "in vivo", arterial infusions of adenosine 5'-triphosphate (ATP) and vasoactive intestinal polypeptide (VIP) not only evoked gastric inhibitory motor responses, but influenced the tone of the stomach. ATP increased and VIP decreased the gastric tone. The effects on the tone appeared strictly related to the inhibitory motility patterns elicited in the stomach by the two non-adrenergic, non-cholinergic neurotransmitters.

Depression by fenoprofen of the rebound contractions' elicited by vagal stimulation and arterial infusion of ATP in the rabbit stomach in vivo.

In the atropine-treated rabbit, vagal stimulation, arterial infusion of ATP or vasoactive intestinal peptide (VIP) caused gastric relaxation. At the end of either vagal stimulation or ATP infusion, but not after VIP, the gastric inhibitory responses were abruptly interrupted by 'rebound contractions'. Administration of fenoprofen depressed or abolished the rebound contraction, thus transforming the brisk relaxant response, elicited by vagal stimulation or ATP, into long-lasting relaxation. Indomethacin depressed the rebound contractions only at high doses and this effect was not always reproducible.

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 alpha,beta-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.

[The presence of purinergic, quinacrine-positive neurons in the rabbit stomach]. / Sulla presenza di neuroni purinergici, chinacrino-positivi nello stomaco di coniglio.

Inhibitory non-adrenergic non-cholinergic efferent nerves are activated in the stomach of the rabbit by electrical vagal stimulation (1). Aim of the present research is to ascertain, in the rabbit stomach, by means of quinacrine fluorescence technique (6) the presence of quinacrine-positive cells and nerve fibers which are thought to be non-adrenergic non-cholinergic inhibitory nerves of gastric motility. A population of neurons showing a high affinity for quinacrine was revealed by fluorescence microscopy in the myenteric plexus of the rabbit stomach.