Glucagon-like peptide 2 counteracts the mucosal damage and the neuropathy induced by chronic treatment with cisplatin in the mouse gastric fundus.

BACKGROUND: Glucagon-like peptide-2 (GLP-2) is a pleiotropic hormone synthesized and secreted by the enteroendocrine 'L' cells able to exert intestine-trophic and anti-inflammatory effects. The antineoplastic drug cisplatin causes gastrointestinal alterations with clinical symptoms (nausea and vomiting) that greatly affect the therapy compliance. Experimentally, it has been reported that chronic cisplatin treatment caused mucosal damage and enteric neuropathy in the rat colon. METHODS: We investigated, through a combined immunohistochemical and functional approach, whether [Gly(2) ]GLP-2, a GLP-2 analog, was able to counteract the detrimental effects of long-term cisplatin administration in the mucosa and myenteric neurons of mouse gastric fundus. KEY RESULTS: Morphological experiments showed a reduction in the epithelium thickness in cisplatin-treated mice, which was prevented by [Gly(2) ]GLP-2 co-treatment. Immunohistochemistry demonstrated that cisplatin caused a significant decrease in myenteric neurons, mainly those expressing neuronal nitric oxide synthase (nNOS), that was prevented by [Gly(2) ]GLP-2 co-treatment. In the functional experiments, [Gly(2) ]GLP-2 co-treatment counteracted the increase in amplitude of the neurally induced contractions observed in strips from cisplatin-treated animals. The NO synthesis inhibitor L-N(G) -nitro arginine caused an increase in amplitude of the contractile responses that was greater in preparations from cisplatin+[Gly(2) ]GLP-2 treated mice compared to the cisplatin-treated ones. CONCLUSIONS & INFERENCES: The results demonstrate that in cisplatin long-term treated mice [Gly(2) ]GLP-2 is able to counteract both the mucosal gastric fundus damage, by preventing the epithelium thickness decrease, and the neuropathy, by protecting the nNOS neurons. Taken together, the present data suggest that [Gly(2) ]GLP-2 could represent an effective strategy to overcome the distressing gastrointestinal symptoms present during the anti-neoplastic therapy.

Relaxin exerts two opposite effects on mechanical activity and nitric oxide synthase expression in the mouse colon.

The hormone relaxin exerts a variety of functions on the smooth muscle of reproductive and nonreproductive organs, most of which occur through a nitric oxide (NO)-mediated mechanism. In the stomach and ileum, relaxin causes muscle relaxation by modulating the activity and expression of different nitric oxide synthase (NOS) isoforms region-dependently. Nothing is known on the effects of relaxin in the colon, the gut region expressing the highest number of neuronal (n) NOSß-immunoreactive neurons and mainly involved in motor symptoms of pregnancy and menstrual cycle. Therefore, we studied the effects of relaxin exposure in the mouse proximal colon in vitro evaluating muscle mechanical activity and NOS isoform expression. The functional experiments showed that relaxin decreases muscle tone and increases amplitude of spontaneous contractions; the immunohistochemical results showed that relaxin increases nNOSß and endothelial (e) NOS expression in the neurons and decreases nNOSα and eNOS expression in the smooth muscle cells (SMC). We hypothesized that, in the colon, relaxin primarily increases the activity and expression of nNOSß and eNOS in the neurons, causing a reduction of the muscle tone. The downregulation of nNOSα and eNOS expression in the SMC associated with increased muscle contractility could be the consequence of continuous exposue of these cells to the NO of neuronal origin. These findings may help to better understand the physiology of NO in the gastrointestinal tract and the role that the "relaxin-NO" system plays in motor disorders such as functional bowel disease.

Endocannabinoids modulate non-adrenergic, non-cholinergic inhibitory neurotransmission in strips from the mouse gastric fundus.

AIM: To investigate the effects of endocannabinoids on non-adrenergic, non-cholinergic (NANC) relaxant responses in gastric strips from mice. METHODS: Gastric longitudinal strips from the fundus region were mounted in organ baths for isometric recording. RESULTS: In carbachol-precontracted strips, electrical field stimulation (EFS) elicited tetrodotoxin (TTX)-sensitive fast nitrergic relaxant responses that were followed, at the highest stimulation frequency, by sustained relaxations. The latter were abolished by α-chymotrypsin. Anandamide caused a TTX-sensitive relaxation that was abolished by α-chymotrypsin but unaffected by the nitric oxide (NO) synthesis inhibitor, Nω-nitro-L-arginine (L-NNA). Anandamide reduced the amplitude of EFS-induced fast relaxations, whereas increased that of sustained ones. Relaxation to the nicotinic receptor agonist dimethylphenyl piperazinium iodide (DMPP) was decreased in amplitude by either anandamide or L-NNA, whereas, surprisingly, it was increased by α-chymotrypsin and abolished by L-NNA plus α-chymotrypsin. Relaxation to vasoactive intestinal polypeptide (VIP) was not influenced by anandamide or L-NNA and was abolished by α-chymotrypsin. Following VIP desensitization, fast relaxant responses to EFS were reduced and the sustained ones abolished. The CB1 receptor antagonist AM251 increased, only at the highest stimulation frequency, the amplitude of the EFS-induced fast relaxation and reduced the sustained one. AM251 increased the response to DMPP and abolished that to anandamide. The CB2 receptor antagonist AM630 had no effects. CONCLUSION: These results indicate that endocannabinoids modulate, via prejunctional CB1 receptors, the NANC peptidergic neurotransmission that, in turn, affects the nitrergic one.

Relaxin counteracts the altered gastric motility of dystrophic (mdx) mice: functional and immunohistochemical evidence for the involvement of nitric oxide.

Impaired gastric motility ascribable to a defective nitric oxide (NO) production has been reported in dystrophic (mdx) mice. Since relaxin upregulates NO biosynthesis, its effects on the motor responses and NO synthase (NOS) expression in the gastric fundus of mdx mice were investigated. Mechanical responses of gastric strips were recorded via force displacement transducers. Evaluation of the three NOS isoforms was performed by immunohistochemistry and Western blot. Wild-type (WT) and mdx mice were distributed into three groups: untreated, relaxin pretreated, and vehicle pretreated. In strips from both untreated and vehicle-pretreated animals, electrical field stimulation (EFS) elicited contractile responses that were greater in mdx than in WT mice. In carbachol-precontracted strips, EFS induced fast relaxant responses that had a lower amplitude in mdx than in WT mice. Only in the mdx mice did relaxin depress the amplitude of the neurally induced excitatory responses and increase that of the inhibitory ones. In the presence of L-NNA, relaxin was ineffective. In relaxin-pretreated mdx mice, the amplitude of the EFS-induced contractile responses was decreased and that of the fast relaxant ones was increased compared with untreated mdx animals. Responses to methacholine or papaverine did not differ among preparations and were not influenced by relaxin. Immunohistochemistry and Western blotting showed a significant decrease in neuronal NOS expression and content in mdx compared with WT mice, which was recovered in the relaxin-pretreated mdx mice. The results suggest that relaxin is able to counteract the altered contractile and relaxant responses in the gastric fundus of mdx mice by upregulating nNOS expression.

Orexins and gastrointestinal functions.

Orexin A (OXA) and orexin B (OXB) are recently discovered neuropeptides that appear to play a role in various distinct functions such as arousal and the sleep-wake cycle as well as on appetite and regulation of feeding and energy homeostasis. Orexins were first described as neuropeptides expressed by a specific population of neurons in the lateral hypothalamic area, a region classically implicated in feeding behaviour. Orexin neurons project to numerous brain regions, where orexin receptors have been shown to be widely distributed: both OXA and OXB act through two subtypes of receptors (OX1R and OX2R) that belong to the G protein-coupled superfamily of receptors. Growing evidence indicates that orexins act in the central nervous system also to regulate gastrointestinal functions: animal studies have indeed demonstrated that centrally-injected orexins or endogenously released orexins in the brain stimulates gastric secretion and influence gastrointestinal motility. The subsequent identification of orexins and their receptors in the enteric nervous system (including the myenteric and the submucosal plexuses) as well as in mucosa and smooth muscles has suggested that these neuropeptides may also play a local action. In this view, emerging studies indicate that orexins also exert region-specific contractile or relaxant effects on isolated gut preparations. The aim of the proposed review is to summarize both centrally- and peripherally-mediated actions of orexins on gastrointestinal functions and to discuss the related physiological role on the basis of the most recent findings.

Relaxin and nitric oxide signalling.

The peptide hormone relaxin (RLX) has been shown to exert a variety of functions in both reproductive and non-reproductive tissues. The molecular mechanism of RLX on its target cells appears to involve multiple intracellular signalling systems, including the nitric oxide (NO) pathway. NO is an ubiquitous molecule synthesised from L-arginine under the catalytic action of different nitric oxide synthase (NOS) isoforms and its altered production has been reported to be involved in several diseases. RLX has been demonstrated to promote NO biosynthesis by up-regulating NOS expression; its influence on the different NOS appears to depend on the cell type studied. In addition to its physiological roles, RLX has been postulated as a potential therapeutic agent in several diseases. In particular, based on its property to promote NO biosynthesis, RLX may be regarded as a therapeutic tool in diseases characterized pathogenically by an impaired NO production. The aim of the present mini-review is to summarize and discuss the pathophysiological actions of RLX, strictly related to its ability to activate the endogenous NO pathway in reproductive and non-reproductive target organs.

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.

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.

Inducible nitric oxide synthase expression in vascular and glomerular structures of human chronic allograft nephropathy.

Nitric oxide (NO) plays an important role in the cytotoxic mechanisms responsible for acute renal allograft rejection, where macrophages produce high levels of inducible nitric oxide synthase (iNOS). By contrast, both the source and the role of NO in chronic allograft nephropathy (CAN) are still unclear. In this study, the expression of iNOS mRNA and protein was assessed in the kidneys of patients with graft failure due to chronic rejection. As controls, kidney specimens were obtained from patients undergoing nephrectomies for primary renal tumours, and from patients suffering from IgA nephropathy or mesangial-proliferative glomerulonephritis. In normal kidneys, iNOS production was absent or limited to a low signal, while it was found only in the inflammatory infiltrate of kidneys affected by glomerulonephritis, as assessed by immunohistochemistry and in situ hybridization. In contrast, in CAN, iNOS protein was localized not only in inflammatory cells, but also in vascular, glomerular, and, more rarely, tubular structures. Accordingly, in situ hybridization localized iNOS mRNA in both macrophages and lymphocytes, as well as in vascular structures and glomeruli. Double immunostaining for iNOS and a-smooth muscle actin (a-SMA) or von Willebrand factor (vWf) revealed that smooth muscle cells were the main vascular source of iNOS, while both mesangial and inflammatory cells were immunostained at the glomerular level. These data demonstrate that macrophages and lymphocytes are not the only source of iNOS mRNA and protein in human CAN. Vascular smooth muscle and mesangial cells also synthesize iNOS, raising the question of heterogeneous regulation and function of iNOS in this disease.

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.

Possible involvement of neuropeptidergic sensory nerves in alopecia areata.

Alopecia areata (AA) is a dermatosis involving the sudden occurrence of bald patches on the scalp. Although the aetiology is unknown, experimental data indicate that cutaneous microcirculation plays an important role. The skin is richly innervated by neuropeptidergic sensory nerves that help regulate microvascular circulation. This study shows a reduction of cutaneous levels of substance P and calcitonin gene-related peptide (CGRP) but not of vasoactive intestinal polypeptide in scalp biopsies from patients with AA. Laser-Doppler flowmetry was used to study microcirculation of the scalp. Results indicate that patients with AA have lower basal blood flow and greater vasodilatation following intradermal CGRP injection than control subjects. A vascular hyper-reactivity to vasodilatatory substances such as neuropeptides, probably because of the lack of these substances, is hypothesized.

T cells and cytokines in Crohn's disease.

Recent findings indicate that activated T lymphocytes, showing restricted T-cell receptor repertoire and a Th1-like profile of cytokine production, are responsible for macrophage activation and release of inflammatory cytokines, toxic oxygen metabolites and nitric oxide, which initiate and maintain the transmural intestinal inflammation in Crohn's disease. A critical event in the promotion of Th1-type response at gut level may involve up-regulation of IL-12 production and the breakdown of tolerance against the intestinal flora.

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.