Changes in enteric neurone phenotype and intestinal functions in a transgenic mouse model of enteric glia disruption.

AIMS: The influence of enteric glia on the regulation of intestinal functions is unknown. Our aim was to determine the phenotype of enteric neurones in a model of glia alterations and the putative changes in intestinal motility and permeability. METHODS: Transgenic mice expressing haemagglutinin (HA) in glia were used. Glia disruption was induced by injection of activated HA specific CD8+ T cells. Control mice consisted of non-transgenic littermates injected with activated HA specific CD8+ T cells. Immunohistochemical staining for choline acetyltransferase (ChAT), substance P (SP), vasoactive intestinal peptide (VIP), and nitric oxide synthase (NOS) was performed on jejunal submucosal plexus (SMP) and myenteric plexus (MP). Neurally induced jejunal muscle activity was characterised in vitro. Gastrointestinal transit and paracellular permeability were measured using fluorescein isothiocyanate-dextran markers. RESULTS: CD3 positive T cells infiltrates were observed in the MP of transgenic mice. In the SMP, the proportions of VIP and SP positive neurones decreased in transgenic mice compared with control mice. ChAT remained unchanged. In the MP, the proportions of ChAT and NOS positive neurones increased and decreased, respectively, in transgenic mice. In contrast, VIP and SP remained unchanged. Neurally mediated jejunal relaxation was lower in transgenic mice than in controls. This relaxation was reduced by NG-nitro-L-arginine methyl ester in control mice but not in transgenic mice. Gastrointestinal transit was delayed and intestinal permeability increased in transgenic mice compared with control mice. CONCLUSION: Glia disruption induces changes in the neurochemical coding of enteric neurones, which may partly be responsible for dysfunctions in intestinal motility and permeability.

Projections of excitatory and inhibitory motor neurones to the circular and longitudinal muscle of the guinea pig colon.

The aim of this study was to identify myenteric pathways to the circular and longitudinal muscle of the guinea pig proximal colon. To identify excitatory and inhibitory muscle motoneurones, we applied the neuronal retrograde tracer DiI onto the circular or longitudinal muscle layer and performed additional immunohistochemistry for nitric oxide synthase (NOS) and choline acetyltransferase (ChAT). On average 166 +/- 81 circular muscle motoneurones (CMMN) and 100 +/- 74 longitudinal muscle motoneurones (LMMN) were labelled by DiI tracing. Myenteric pathways innervating the muscle were either ascending (DiI-labelled neurones with oral projections) or descending (DiI-labelled neurones with anal projections). The circular muscle was preferentially innervated by ascending pathways (66.0 +/- 9.1%). Most ascending CMMN were ChAT-positive (87.2 +/- 8.5%), whereas descending CMMN were mainly NOS-positive (82.3 +/- 14.6%). Most ascending (62.2 +/- 11.1%) and descending (82.0 +/- 12.5%) CMMN had circumferential projection preferences (circumferential projections were longer than projections along the longitudinal gut axis). In contrast to the polarised projections to the circular muscle, the longitudinal muscle was equally innervated by ascending (46.2 +/- 15.1%) and descending (53.9 +/- 15.1%) neurones. Ascending and descending pathways to the longitudinal muscle consisted predominantly of ChAT-positive neurones (98.1 +/- 1.9% and 68.0 +/- 8.5%, respectively), and both pathways had prominent longitudinal projection preferences. Only 25.5% of the descending LMMN were NOS-positive. In conclusion, the circular muscle in the proximal colon is innervated by descending inhibitory (NOS-positive neurones) and ascending excitatory (ChAT-positive neurones) pathways. In contrast, the longitudinal muscle is primarily innervated by ascending and descending excitatory motoneurones, and only a small proportion of the descending pathway consisted of inhibitory motoneurones.

Interleukin-8 increases acetylcholine response of rat intestinal segments.

BACKGROUND: Interleukin-8 (IL-8) is a pro-inflammatory cytokine highly expressed in inflammatory bowel diseases, but whose effects on intestinal motility are unknown. AIM: To characterize the role of IL-8 in the contraction of rat intestinal segments. METHODS: Contractile response to acetylcholine (ACh 10-6 M) in terminal ileal segments (including mucosa) from Wistar rats was measured before and after incubation (15, 30, 60 or 90 min) with IL-8 (1 ng/mL), and after 60 min of incubation with different doses of IL-8 (0, 0.1, 0.5, 1, 10 and 100 ng/mL). The effects of blocking neural transmission with tetrodotoxin (TTX) and inhibiting protein synthesis (cycloheximide) were tested. The contractile response of longitudinal muscle-myenteric plexus preparations (i.e. without mucosa) was measured after 60 min of incubation with 0.1 and 1 ng/mL of IL-8. RESULTS: IL-8 increased ileal contraction induced by ACh 10(-6) M. This augmentation was significant after 60 min of incubation (58%, P=0.01) and persisted after 90 min (18%, P=0.04). A 60-min incubation period showed a dose-related effect, beginning at 0.5 ng/mL (30%, P=0.003) and reaching a peak at 1 ng/mL (58%, P=0.01). The same effect was also observed on colonic segments. TTX did not affect the IL-8 increase of ACh-induced contractions, which was completely abolished by cycloheximide. IL-8 had no significant effect on longitudinal muscle-myenteric plexus preparations. CONCLUSION: In vitro, IL-8 increases contractile response of the ileum to ACh in a dose-dependent manner. This effect is not neurally mediated, but seems to involve protein synthesis by intestinal mucosa.

Vasoactive intestinal peptide is involved in the inhibitory effect of interleukin-1 beta on the jejunal contractile response induced by acetylcholine.

UNLABELLED: Although previous studies have shown that interleukin-1 beta (IL-1 beta) decreases acetylcholine (ACh)-induced intestinal contraction by an action on the enteric nervous system, the neuromediator(s) involved are still unknown. AIM: To determine the role of nitric oxide (NO), vasoactive intestinal peptide (VIP) and/or adenosine triphosphate (ATP) in mediating this inhibitory effect. METHODS: The effects of NO synthase inhibitors, VIP and ATP antagonists on motor response to the ACh were investigated before and after 90-min exposure of a rat preparation of jejunal longitudinal muscle-myenteric plexus to IL-1 beta. NG-nitro-L-arginine methyl ester, NG-nitro-L-arginine and NG-monomethyl-L-arginine were used to inhibit NO synthase, VIP (10-28) and [D-p-Cl-Phe6, Leu17] VIP to block VIP receptors, and suramin to block ATP receptors. RESULTS: NO synthase inhibitors failed to block the inhibitory effect of IL-1 beta on ACh-contracted jejunum smooth muscle. Suramin also failed to affect IL-1 beta-induced inhibition, whereas VIP antagonists abolished it. Moreover, the action of IL-1 beta was partly reproduced by VIP. CONCLUSIONS: While neither NO nor ATP accounts for the inhibitory effect of IL-1 beta on ACh-contracted jejunum, VIP seems to be a key-mediator of this effect.

Altered myoelectrical activity in noninflamed ileum of rats with colitis induced by trinitrobenzene sulphonic acid.

Changes in gastric emptying and orocaecal transit time in patients with ulcerative colitis suggest that disturbances in gut motility may not be restricted to inflamed sites. This study sought to characterize changes in the motility of noninflamed ileum in a rat colitis model and to explore the mechanism(s) potentially involved. The myoelectrical activity of the ileum was recorded in rats with trinitrobenzene sulphonic acid (TNBS)-induced colitis. The degree of ileal and colonic inflammation was assessed by quantification of macroscopic damage and myeloperoxidase activity (MPO). The effect on ileal motility of pretreatment with atropine, indomethacin and NG-nitro-L-arginine-methyl ester (L-NAME) was investigated. TNBS-induced inflammation was restricted to the distal colon, as evidenced by morphological scores and MPO. Colitis was associated with increased frequency of ileal migrating motor complexes, characterized mainly by a decrease in the duration of phases I and III. The occurrence of ileal giant migrating complexes remained unchanged. The myoelectrical changes observed in the ileum persisted after treatment with atropine, indomethacin and L-NAME. Distal colitis is associated with abnormal myoelectrical activity in the noninflamed ileum of rats. Neither acetylcholine nor prostaglandins and nitric oxide seem to be involved.

Elevated plasma leptin concentrations in early stages of experimental intestinal inflammation in rats.

BACKGROUND: Although leptin, an adipocyte derived hormone which regulates food intake and energy balance, is released after injections of tumour necrosis factor (TNF) and interleukin 1, plasma concentrations have not been characterised in chronic inflammation. Leptin may contribute to the anorexia and body weight loss associated particularly with the acute stages of inflammatory bowel disease. AIMS: To investigate plasma leptin concentrations during the time course of intestinal inflammation in different animal models. METHODS: Plasma leptin was measured at different time points in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis, indomethacin induced ileitis, or endotoxic shock caused by lipopolysaccharide (LPS). Systemic TNF-alpha was also measured during acute inflammation. RESULTS: Plasma leptin concentrations increased fourfold eight hours after induction of TNBS colitis (p<0.0001) and twofold after administration of ethanol alone (p<0.02). Plasma leptin responses throughout the first post-treatment day were correlated with myeloperoxidase activity and gross damage scores. Similar leptin overexpression was observed in indomethacin induced ileitis and in rats with endotoxic shock. Plasma concentrations were lower in TNBS treated rats than in controls on day 5 before reaching a similar concentration on day 14. Anorexia and body weight loss were observed during the first four days post-TNBS. A significant increase in systemic TNF-alpha was only detected in LPS treated rats. CONCLUSION: Elevated plasma leptin concentrations, correlated with the degree of inflammation and associated with anorexia, were induced in rats during the early stages of experimental intestinal inflammation but proved transient; this might account for discrepancies in recent results concerning concentrations in patients with inflammatory bowel diseases.

Digestive and metabolic effects of potato and maize fibres in human subjects.

The physiological effects of dietary fibres in humans are due to their physico-chemical properties. However, it is difficult to predict these effects simply by measuring certain characteristics in vitro. Studies in human subjects are still required to assess the effectiveness of new substrates. The aim of the present study in healthy human subjects was to evaluate the effects of two novel fibres, potato (PF) and maize (MF), on fasting and postprandial blood concentrations of carbohydrate and lipid metabolites as well as on stool output and transit time. The chemical composition, water-binding capacity (WBC) and fermentative properties of the fibres were also characterized in order to determine their possible involvement in digestive and metabolic effects. Stools, as well as breath and blood samples, were collected after consumption for 1 month of either a basal diet (control) or a basal diet supplemented with fibre (15 g/d). MF resisted fermentation better than PF and had lower digestibility. However, both fibres increased faecal output of dry matter, neutral sugars and water. There was an inverse relationship between stool weight and orofaecal transit time, although only MF significantly reduced transit time. Orocaecal transit was lengthened by PF, probably because of its high WBC. PF ingestion also decreased postprandial plasma levels of total and esterified cholesterol but had no effect on fasting concentrations. In contrast, MF lowered fasting cholesterolaemia and increased free:esterified cholesterol. These particular physiological and fermentative properties suggest that PF and MF would be suitable ingredients in a healthy diet.

Effects of short-chain fatty acids on gastrointestinal motility.

Besides their action on gut morphology and function, short-chain fatty acids (SCFAs), produced by bacterial fermentation of carbohydrates in the colon, influence gastrointestinal motility. As they are not present in the stomach and proximal small intestine, SCFAs do not directly affect motility of these segments. However, caecal infusion of SCFAs as well as colonic fermentation of lactulose induce a relaxation of the proximal stomach in humans, indicating that SCFAs can affect motility at a distance from their site of production. Moreover, this suggests that SCFAs may be involved in the so-called "ileocolonic brake', i.e. the inhibition of gastric emptying by nutrients reaching the ileo-colonic junction. In the terminal ileum, where their concentration may increase following a colo-ileal reflux, SCFAs stimulate contractions and shorten ileal emptying, which may protect ileal mucosa against the potentially harmful effects of the reflux of colonic contents. Although SCFAs are produced and concentrated in the colon, their action on motility of this organ is not clearly understood and may depend on concentration, molecular structure of the acids, responsiveness of the colonic segments and animal species. The mechanisms of action of SCFAs on gastrointestinal motility are not completely elucidated. They may involve systemic humoral and neural pathways as well as local reflexes and myogenic responses.

Inhibition of acetylcholine induced intestinal motility by interleukin 1 beta in the rat.

BACKGROUND/AIMS: The fact that raised interleukin 1 beta (IL 1 beta) concentrations have been found in the colonic mucosa of rats with experimentally induced colitis and of patients with inflammatory bowel disease indicates that this cytokine may participate in the disturbed intestinal motility seen during inflammatory bowel disease. This study investigated whether IL 1 beta could change the contractility of (a) a longitudinal muscle-myenteric plexus preparation from rat jejunum, ileum, and colon and (b) isolated jejunal smooth muscle cells. METHODS: Isometric mechanical activity of intestinal segments was recorded using a force transducer. Moreover, smooth muscle cell length was measured by image analysis. RESULTS: Although IL 1 beta did not affect jejunal, ileal, and colonic basal contractility, it significantly reduced contractile response to acetylcholine (ACh). This significant inhibition was seen only after 90 or 150 minutes of incubation with IL 1 beta. Pretreatment with cycloheximide blocked IL 1 beta induced inhibition of ACh stimulated jejunal contraction, suggesting that a newly synthesised protein was involved in the effect. NW-nitro-L-arginine (a nitric oxide synthase inhibitor) did not prevent the inhibition induced by IL 1 beta. Blocking neural transmission with tetrodotoxin abolished the IL 1 beta effect on jejunal contractile activity, whereas IL 1 beta had no effect on isolated and dispersed smooth muscle cells. CONCLUSIONS: IL 1 beta inhibits ACh induced intestinal contraction and this inhibitory effect involves protein synthesis but is independent of nitric oxide synthesis. This effect does not involve a myogenic mechanism but is mediated through the myenteric plexus.

In vitro contractile effects of short chain fatty acids in the rat terminal ileum.

Short chain fatty acids (SCFAs), produced in the gut by bacterial fermentation of carbohydrates, change intestinal motility by mechanisms as yet unknown. This study examined the mechanism(s) of action of SCFAs on contractility using isolated rat terminal ileum segments and isolated ileal smooth muscle cells. Strip contractions were recorded under isometric conditions. Intracellular calcium concentration ([Ca2+]i) was measured in single cells loaded with indo-1 penta-acetoxymethyl ester (indo-1 AM). SCFAs (10(-9) to 10(-2) mol/l) induced concentration dependent contractions. The effect was not different among the individual SCFAs. Exogenous acids (namely tartaric and citric acids) caused similar responses as SCFAs, whereas sodium acetate had no effect. The contraction was not blocked by tetrodotoxin, atropine or hexamethonium, showing that it was not mediated through a cholinergic pathway. Moreover, removal of the mucosa or addition of procaine (a local anaesthetic) to the bath did not change the SCFA induced contraction, while verapamil (a calcium-channel antagonist) completely suppressed it. In addition, application of SCFAs to isolated ileal myocytes evoked peaks in [Ca2+]i inhibited by D 600 (a blocker of voltage dependent calcium channels). Taken together, these results suggest that the contractile response stimulated by SCFAs in the rat terminal ileum could result from an acid sensitive calcium dependent myogenic mechanism.