Changes of excitatory and inhibitory neurotransmitters in the colon of rats underwent to the wrap partial restraint stress.

BACKGROUND: Animal models proposed to reproduce some of the human irritable bowel syndrome (IBS) symptoms are based on the hypothesis that psychosocial stressors play a pivotal role in the IBS etio-pathology. We investigated the wrap restraint stress (WRS) model with the aim to analyze the morphological changes of the entire colonic wall of these animals that showed some of the human IBS symptoms such as visceral hypersensitivity. METHODS: Male Wistar rats were used and WRS was maintained for 2 h. Abdominal contractions (AC) were recorded in the colon-rectum by balloon distension. Fecal pellets were quantitated. Colonic specimens were examined by routine histology, immunohistochemistry and western blot. KEY RESULTS: WRS animals were characterized by: (i) increase in AC number and fecal pellets mean weight; (ii) clusters of mononucleated cells, increase in eosinophilic granulocytes and mast cells in the mucosa; (iii) increase in CGRP-immunoreactive (IR) nerve fibers in the lamina propria; (iv) decrease in myenteric NK1r-IR and nNOS-IR neurons and in submucous nNOS-IR neurons; (v) decrease in SP-IR nerve fibers in the muscle wall; (vi) reduction in S100ß-IR glia in the entire colonic wall; (vii) increase in CRF1r-IR myenteric neurons; (viii) no change in ChAT-IR neurons, smooth muscle cells and interstitial cells of Cajal. CONCLUSIONS AND INFERENCES: The present results support the consistency of the WRS as a potential model where part of the human IBS signs and symptoms are reproduced. The changes in glial cells and in excitatory and inhibitory neurotransmitters might represent the substrate for the dysmotility and hypersensitivity.

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.

Changes in nitrergic and tachykininergic pathways in rat proximal colon in response to chronic treatment with otilonium bromide.

BACKGROUND: Otilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well-characterized, little is known about the effects of chronic administration of OB on enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function. METHODS: Adult Sprague-Dawley rats were treated with OB in drinking water at a dose of 2 mg/kg for 30 days. The colons of OB-treated and age-matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology. KEY RESULTS: Using immunohistochemistry, chronic OB administration did not change total neuron number, assessed by anti-Hu IR, but resulted in a significant increase in NK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. Chronic OB administration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonic ICC, detected by Kit IR, were not altered nor were slow waves or smooth muscle membrane potential. CONCLUSIONS & INFERENCES: Chronic treatment with OB caused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.

Chronic treatment with otilonium bromide induces changes in L-type Ca²âº channel, tachykinins, and nitric oxide synthase expression in rat colon muscle coat.

BACKGROUND: Otilonium bromide (OB) is a quaternary ammonium derivative used for the treatment of intestinal hypermotility and is endowed with neurokinin2 receptor (NK2r) antagonist and Ca²âº channel blocker properties. Therefore, the possibility that OB might play a role in the neurokinin receptor/Substance-P/nitric oxide (NKr/SP/NO) circuit was investigated after chronic exposition to the drug. METHODS: Rats were treated with OB 2-20 mg kg⁻¹ for 10 and 30 days. In the proximal colon, the expression and distribution of muscle NOsynthase 1 (NOS1), NK1r, NK2r, SP and Cav 1.2 subunit (for L-type Ca²âº channel) and the spontaneous activity and stimulated responses to NK1r and NK2r agonists were investigated. KEY RESULTS: Immunohistochemistry showed a redistribution of NK1r and L-type Ca²âº channel in muscle cells with no change of NK2r at 30 days, a significant increase in muscle NOS1 expression at 10 days and a significant decrease in the SP content early in the ganglia and later in the intramuscular nerve fibers. Functional studies showed no change in spontaneous activity but a significant increase in maximal contraction induced by NK1r agonist. CONCLUSIONS & INFERENCES: Chronic exposition to OB significantly affects the NKr/SP/NO circuit. The progressive decrease in SP-expression might be the consequence of the persistent presence of OB, the increase of NOS1 expression in muscle cells at 10 days in an attempt to guarantee an adequate NO production, and, at 30 days, the redistribution of the L-type Ca²âº channel and NK1r as a sign to compensate the drug channel block by re-cycling both of them. The physiological data suggest NK1r hypersensitivity.

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.

GLP-2 receptor expression in excitatory and inhibitory enteric neurons and its role in mouse duodenum contractility.

BACKGROUND: Glucagon-like peptide 2 (GLP-2), a nutrient-responsive hormone, exerts various actions in the gastrointestinal tract that are mediated by a G-protein coupled receptor called GLP-2R. A little information is available on GLP-2R expression in enteric neurons and nothing on the interstitial cells of Cajal (ICC). METHODS: We investigated presence and distribution of the GLP-2R in the mouse duodenum by immunohistochemistry and the potential motor effects of GLP-2 on the spontaneous and neurally evoked mechanical activity. KEY RESULTS: The GLP-2R was expressed by the myenteric and submucosal neurons. Labelling was also present in nerve varicosities within the circular muscular layer and at the deep muscular plexus (DMP). No immunoreactive nerve fiber was seen within the longitudinal muscle layer. The GLP-2R-positive neurons were either excitatory (SP- and choline-acetyltransferase-positive) or inhibitory (vasoactive intestinal polypeptide and nNOS-positive). The ICC, both at the myenteric plexus and at the DMP, never expressed GLP-2R but, especially those at the DMP, were surrounded by GLP-2R-positive nerve varicosities co-expressing either excitatory or inhibitory neurotransmitters. Quantitative analysis demonstrated a consistent prevalence of GLP-2R on the excitatory pathways. In agreement, the functional results showed that the administration of GLP-2 in vitro caused decrease of the spontaneous contractions mediated by nitric oxide release and reduction of the evoked cholinergic contractions. CONCLUSIONS & INFERENCES: The present findings indicate that the GLP-2R is expressed by inhibitory and excitatory neurons, the GLP-2 inhibits the muscle contractility likely decreasing cholinergic neurotransmission and increasing nitric oxide production, and this effect is possibly mediated by the ICC-DMP recruitment.

Effect of otilonium bromide and ibodutant on the internalization of the NK2 receptor in human colon.

BACKGROUND: The present aim was to study the modulation of NK2 receptor internalization by two compounds, the spasmolytic otilonium bromide (OB) endowed with NK2 receptor antagonistic properties and the selective NK2 receptor antagonist ibodutant. METHODS: Full-thickness human colonic segments were incubated in the presence of OB (0.1-10 µmol L(-1)) or ibodutant (0.001-0.1 µmol L(-1)), with or without the NK2 receptor selective agonist [ßAla8]NKA(4-10) and then fixed in 4% paraformaldehyde. Cryosections were processed for NK2 receptor immunohistochemical revelation. Quantitative analysis evaluated the number of the smooth muscle cells that had internalized the NK2 receptor. KEY RESULTS: Immunohistochemistry revealed that in basal condition, the NK2 receptor was internalized in about 23% of total smooth muscle cells. The exposure to the selective NK2 receptor agonist induced internalization of the receptor in more than 77% of the cells. Previous exposure to both OB or ibodutant, either alone or in the presence of the agonist, concentration-dependently reduced the number of the cells with the internalized receptor. CONCLUSIONS & INFERENCES: Both OB and ibodutant antagonize the internalization of the NK2 receptor in the human colon. As NK2 receptors are the predominant receptor mediating spasmogenic activity of tachykinins on enteric smooth muscle, we hypothesize that the antagonistic activity found for both OB and ibodutant should play a specific therapeutic role in gut diseases characterized by hypermotility.

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.

Peripheral motor action of glucagon-like peptide-1 through enteric neuronal receptors.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived peptide expressed in the enteroendocrine-L cells of small and large intestine and released in response to meal ingestion. Glucagon-like peptide-1 exerts inhibitory effects on gastrointestinal motility through vagal afferents and central nervous mechanisms; however, no data is available about a direct influence on the gastrointestinal wall. Our aim was to investigate the effects of GLP-1 on the spontaneous and evoked mechanical activity of mouse duodenum and colon and to identify the presence and distribution of GLP-1 receptors (GLP-1R) in the muscle coat. METHODS: Organ bath recording technique and immunohistochemistry were used. KEY RESULTS: Glucagon-like peptide-1 (up to the concentration of 1 mumol L(-1)) failed to affect spontaneous mechanical activity. It caused concentration-dependent reduction of the electrically evoked cholinergic contractions in circular smooth muscle of both intestinal segments, without affecting the longitudinal muscle responses. Glucagon-like peptide-1 inhibitory effect was significantly antagonized by exendin (9-39), an antagonist of GLP-1R. In both intestinal preparations, GLP-1 effect was not affected by guanethidine, a blocker of adrenergic neurotransmission, but it was significantly reduced by N(omega)-nitro-l-arginine methyl ester, inhibitor of nitric oxide (NO) synthase. Glucagon-like peptide-1 failed to affect the contractions evoked by exogenous carbachol. Immunohistochemistry demonstrated GLP-1R expression in the enteric neurons. Furthermore, 27% of GLP-1R immunoreactive (IR) neurons in the duodenum and 79% of GLP-1R-IR neurons in the colon, co-expressed nNOS. CONCLUSIONS & INFERENCES: The present results suggest that GLP-1 is able to act in the enteric nervous system by decreasing the excitatory cholinergic neurotransmission through presynaptic GLP-1Rs, which modulate NO release.

Adenosine A2A receptor antagonism increases nNOS-immunoreactive neurons in the striatum of Huntington transgenic mice.

Medium spiny GABAergic projection neurons are progressively lost in Huntington's disease (HD), whereas there is preferential sparing of the few interneurons co-expressing NPY, somatostatin and neuronal nitric oxide synthase. We investigated the effect of the selective adenosine A(2A) receptor antagonist SCH58261 (0.01 mg/kg, acutely and chronically administered i.p.) on nNOS striatal expression and motor impairment in R6/2 transgenic mice in clearly symptomatic phase (10-11-week old). SCH58261 chronically administered increased the number of nNOS-immunoreactive neurons (nNOS-IR) in the striatum of R6/2 mice. No glial activation was detected in the striatum or cortex. SCH58261 also improved walking in the inclined plane test but not motor capability evaluated by the rotarod test. These findings demonstrate for the first time a role of adenosine A(2A) receptors in regulating nNOS expression in the striatum. We suggest that the protective effect of A(2A) antagonism in HD is related to the increase in striatal nNOS-IR neurons.

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.

Altered tachykinergic influence on gastric mechanical activity in mdx mice.

This study investigated whether alterations in gastric activity in dystrophic mdx mouse can be attributed to dysfunctions of tachykinins. Endoluminal pressure was recorded and the expression of neuronal nitric oxide synthase (nNOS), NK1 and NK2 neurokinin receptors was investigated by immunohistochemistry. SR48968, NK2 receptor antagonist, but not SR140333, NK1 receptor antagonist, decreased the tone only in mdx gastric preparations. In the presence of N(omega)-nitro-l-arginine methyl ester (l-NAME), inhibitor of NOS, SR48968 reduced the tone also in normal stomach. [Sar(9), Met(O(2))(11)]-SP, agonist of NK1 receptors, caused tetrodotoxin-sensitive relaxations, antagonized by SR140333 or l-NAME, with no difference in the potency or efficacy between normal and mdx preparations. [beta-Ala(8)]-NKA(4-10), an NK2 receptor agonist, induced SR48968-sensitive contractions in both types of preparations, although the maximal response of mdx tissues was significantly lower than normal preparations. Immunohistochemistry demonstrated a consistent reduction of nNOS and NK2 receptor expression in mdx stomach smooth muscle cells and no change in nNOS and NK1 receptor expression in neurones. In conclusion, in mdx stomach the activation of NK2 receptors plays a role in the development of the tone, associated with a reduced NO production by muscular nNOS. The hypo-responsiveness to NK2 receptors could depend on the reduced expression of these receptors.

Expression of neuronal and inducible nitric oxide synthase in neuronal and glial cells after transient occlusion of the middle cerebral artery.

We presently investigated the time-course of neuronal nitric oxide synthase and inducible nitric oxide synthase expression and content in the rat striatum up to 6 days after ischemia induced by transient middle cerebral artery occlusion, a condition that potentially allows functional recovery, with the aim to identify the cell types expressing these two enzymes and to correlate neuronal nitric oxide synthase and inducible nitric oxide synthase changes in order to verify whether and how these changes are related to tissue damage, motor-sensory performances and survival. Before and after surgery, the animals underwent neurological evaluation. The results demonstrated that the rats with a score > or = 12 at the neurological evaluation 24 h after ischemia showed a significant increase in neuronal nitric oxide synthase-immunoreactive neurones and absence of inducible nitric oxide synthase-immunoreactive cells and survived up to the sixth day; conversely, the rats with a score < 12 at the neurological evaluation 24 h after ischemia showed a progressive significant decrease in neuronal nitric oxide synthase-immunoreactive neurones and appearance of inducible nitric oxide synthase-immunoreactive cells and none of the rats survived up to the sixth day. Microglia cells were activated in both groups but only in the latter did these cells express inducible nitric oxide synthase. Measurement of the infarct area demonstrated that it occupied a similar territory in both groups of rats but in those with a score < 12 the edema was more extended. In conclusion, we demonstrated that a neurotoxic insult such as ischemia can induce neuronal nitric oxide synthase expression in the neurones and that when neuronal nitric oxide synthase-immunoreactive neurones increase in number, microglia activation is less extended, inducible nitric oxide synthase-immunoreactive cells are absent, tissue damage reduced and the rats survive longer. Conversely, when there is a significant decrease of neuronal nitric oxide synthase-immunoreactive neurones, microglia cells are intensely activated, inducible nitric oxide synthase-immunoreactive cells appear and the animal survival is shortened.

Gastroschisis in the rat model is associated with a delayed maturation of intestinal pacemaker cells and smooth muscle cells.

BACKGROUND: A pacemaker system is required for peristalsis generation. The interstitial cells of Cajal (ICC) are considered the intestinal pacemaker, and are identified by expression of the c-kit gene--encoded protein. Gastroschisis is characterized by a severe gastrointestinal dysmotility in newborns. In spite of this clinical picture, few studies have focused on smooth muscle cells (SMC) morphology and none on ICC. Therefore, their morphology has been studied in fetuses at term in the rat model of gastroschisis. METHODS: At 18.5 day's gestation (E18.5), 10 rat fetuses were killed, 10 underwent surgical creation of gastroschisis, and 10 underwent manipulation only. The small intestine of the latter 2 groups was harvested at E21.5. Specimens were processed for H&E, c-kit and actin (alpha smooth muscle antibody [alpha-SMA]) immunohistochemistry, and transmission electron microscopy (TEM). RESULTS: In the controls, SMC were c-kit+ and alpha-SMA+, with labeling intensity increasing by age. At E21.5, some cells around the Auerbach's plexus were more intensely c-kit+, and differentiating ICC were seen under TEM at this level. Gastroschisis fetuses had no c-kit+ cells referable to ICC. In the more damaged loops, SMC were very faintly c-kit+ and alpha-SMA+. Under TEM, there were few differentiated SMC and no presumptive ICC. In the less-damaged loops, SMC were faintly c-kit+ and alpha-SMA+ and had ultrastructural features intermediate between those of E18.5 and E21.5 controls; ICC were very immature. CONCLUSIONS: ICC and SMC differentiation is delayed in gastroschisis with the most damaged loops showing the most incomplete picture. These findings might help in understanding the delayed onset of peristalsis and the variable time-course of the recover seen in babies affected by gastroschisis.

Neuronal differentiation and myenteric plexus organization are delayed in gastroschisis: an immunohistochemical study in a rat model.

Gastroschisis is a malformation due to prenatal rupture of the abdominal wall and evisceration of the midgut. Intestinal loops are shortened, matted, and covered by a peel caused by the harmful effect of the amniotic fluid. Babies born with gastroschisis suffer from gastrointestinal dysmotility. The present aim was to verify whether the myenteric plexus is damaged in a rat model of gastroschisis. In the gastroschisis rat model fetus, the myenteric plexus was not yet organized in the well-defined ganglia and, in the most damaged loops, the neuronal cells were scattered or absent. Immunohistochemistry for alpha-internexin and peripherin (markers of neuronal maturity) gave results similar to those of earlier embryonic ages. These findings indicate a delay in neuronal differentiation and myenteric plexus organization that might play a role in the postnatal dysmotility observed in gastroschisis.

Myenteric neurons and interstitial cells of Cajal of mouse colon express several nitric oxide synthase isoforms.

Information on equipment and subcellular distribution of nitric oxide synthase (NOS) isoforms in myenteric neurons and pacemaker cells (ICC) might help to identify nitric oxide (NO) pathway(s) acting on gastrointestinal motility. In sections of mouse colon labelled with neuronal (n)NOS, endothelial (e)NOS and inducible (i)NOS antibodies, all myenteric neurons co-expressed eNOS and iNOS and a subpopulation of them co-expressed nNOS. ICC co-expressed nNOS and eNOS. In the neurons, nNOS-labeling was intracytoplasmatic, in the ICC at cell periphery. In both cell types, eNOS-labeling was on intracytoplasmatic granules, likely mitochondria. In conclusion, myenteric neurons and ICC co-express several NOS isoforms with specific subcellular distribution. Different nNOS splice variants are presumably present: intracytoplasmatic nNOSbeta and nNOSalpha producing neurogenic NO, plasma membrane-bound nNOSalpha producing ICCgenic NO. eNOS might be implicated in mitochondrial respiration and, in ICC, also in pacemaker activity. Neurons express iNOS also in basal condition.

Alterations of neurokinin receptors and interstitial cells of Cajal during and after jejunal inflammation induced by Nippostrongylus brasiliensis in the rat.

Substance P (SP) and its receptors NK1 and NK2 are widely expressed in the intestinal wall by neurones, interstitial cells of Cajal (ICC) and smooth muscle cells. Changes in SP and/or its NK receptors have been documented during experimental inflammation in animals or inflammatory bowel diseases in humans, but the data concern the acute phase of the inflammatory process. We determined immunohistochemically whether NK receptors and SP were altered in the muscle coat during jejunal inflammation induced by the nematode Nippostrongylus brasiliensis and whether these alterations persisted when inflammation had spontaneously resolved 30 days postinfection. An ultrastructural analysis was also conducted on ICC, nerves and muscle. At day 14, when inflammation peaked, there was a reduction in NK1 receptors in myenteric neurones and in SP-immunoreactive nerve endings. There were also ultrastructural anomalies in synaptic vesicles and NK2 receptor loss in the circular muscle layer. The SP decrease persisted at day 30, whereas neurones and circular muscle cells re-expressed NK1 and NK2 receptors, respectively. The ICC at the deep muscular plexus, located near to the inflammatory site, underwent alterations leading to their complete loss at day 30. These morphological changes are probably associated with impairment in tachykinergic control of jejunal functions leading to the alterations of motility and sensitivity to distension already described in these animals.

Myogenic NOS and endogenous NO production are defective in colon from dystrophic (mdx) mice.

The aim of the present study was to evaluate whether alterations in the distribution and/or function of nitric oxide synthase (NOS) could be involved in the development of the spontaneous mechanical tone observed in colon from dystrophic (mdx) mice. By recording the intraluminal pressure of isolated colon from normal mice, we showed that N(omega)-nitro- L-arginine methyl ester (L-NAME) increased the tone, even in the presence of tetrodotoxin. The effect was prevented by L-arginine, nifedipine, or Ca(2+)-free solution. In colon from mdx mice, L-NAME was ineffective. Immunohistochemistry revealed that the presence and distribution of neuronal (nNOS), endothelial, and inducible NOS isoforms in smooth muscle cells and neurons of colon from mdx mice were the same as in controls. However, the expression of myogenic nNOS was markedly reduced in mdx mice. We conclude that there is a myogenic NOS in mouse colon that can tonically produce nitric oxide to limit influx of Ca(2+) through L-type voltage-dependent channels and modulate the mechanical tone. This mechanism appears to be defective in mdx mice.

Expression of dystrophin in the mouse myenteric neurones.

Dystrophin, a membrane-associated protein, plays relevant roles in cell functions. Its lack or trunkated expression results in Duchenne muscular dystrophy (DMD), a pathology associated with alterations in gastrointestinal motility considered to be neural in origin. No data are available on the presence of dystrophin in myenteric neurones. We labelled mouse myenteric neurones with DYS1-, DYS2-, DYS3-antibodies; staining was located on the perikarya and processes, with no differences in distribution or intensity among the antibodies; the western immunoblot analysis indicated that myenteric neurones express several dystrophin isoforms; anti-dystrophins/anti-neuronal specific enolase double-labeling confirmed that all neurones express dystrophin. Dystrophin in myenteric neurones might play a role in cytoskeletal organization, axonal transport and signal pathways; its lack might cause the intestinal motor abnormalities reported in DMD patients.

Synapse formation during neuron differentiation: an in situ study of the myenteric plexus during murine embryonic life.

Ultrastructural steps characterizing synapse formation in vivo and appearance in neuroblasts of properties suggestive of synaptic function acquisition have scarcely been studied. Synapse formation and proteosynthetic apparatus organization were thus studied under transmission electron microscope in mouse myenteric neurons from embryonic day 12.5 (E12.5) until birth. Expression of Ret and p75(NTR), markers of neural crest cells, as well as that of neuron-specific enolase (NSE), synaptophysin (SY), and synaptosomal-associated protein (SNAP), markers of synaptic function acquisition, were immunohistochemically evaluated. At E12.5 many cells were Ret- and p75(NTR)-immunoreactive (IR), whereas a few were NSE-IR and had neuronal ultrastructural characteristics. Two types of contacts between poorly or nondifferentiated cells and axons of presumed extrinsic (synapse-like contacts) or local (immature synapses) origin were identified, along with SY-IR elements. By E16. 5, many cells had developed a proteosynthetic apparatus, synapse-like contacts were no longer present, and immature synapses were gradually differentiating. Concurrently, there was an increase in NSE-IR cells, some of which were also SNAP-IR, and in SY-IR varicosities. At E18.5, ultrastructurally mature neurons and synapses had increased in number as had NSE-IR and SNAP-IR cells and SY-IR varicosities. These data indicate that 1) one type of contact (synapse-like) is present at E12.5 between very immature cells and presumed vagal fibers, with a possible transient role for the onset of the differentiative process of these cells; and 2) another type of contact (typical synapses) lasts until E18.5, with a similar but long-lasting role that progressively shifts to the classical function (neurotransmission) as the synapse matures and the embryo reaches the day of birth.