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Background Probiotics are live microorganisms that promote gut health and regulate intestinal homeostasis. How probiotics work is incompletely understood, but may involve induction of cell survival pathways (Akt) and stress/MAP kinase-dependent induction of heat shock proteins. The possibility that probiotics might induce autophagy, however, has not been previously explored. Autophagy is believed to have an important role in promoting cell survival under conditions of stress and in clearance of potentially disease-causing intracellular microorganisms. Polymorphisms in autophagy genes have recently been linked to increased risk of human IBD. Methods Intestinal epithelial cells (human colonic Caco2BBE or rat jejunal IEC18) were treated with conditioned media from Bifidobacterium breve (BB-CM) or other intestinal bacteria (Lactobacillus plantarum, Lactobacillus rhamnosus GG). Initially time dependence was determined using 1% CM and subsequently concentration dependence was determined at 30 or 120 min and 24 hours for rapid transduction events (MAP kinase, LC3 conjugation, or Akt activation) versus long lived cellular survival proteins (heat shock protein induction), all assessed by Western blot analysis. Results BB-CM induced autophagy in a timeand concentration-dependent manner. Western blot analysis demonstrated LC3II activation by conjugation to phosphatidylethanolamine by 120 min after BB-CM. BB-CM also activated p38 MAP kinase and ERK1/2, but within 30 min of addition. Both Lactobacillus plantarum and LGG-CM also stimulated LC3, thereby demonstrating stimulation of autophagy. For BB-CM, inhibition of either p38 MAP kinase with SB203580 ort ERK1/2 activation with PD98059 blocked LC3 activation. A wider panel of gram positive and gram negative bacteria were tested only on LC3 activation and while most gram positive bacteria stimulated LC3 activation, most gram negative did not. Conclusions These studies provide evidence that bioactive agents secreted by probiotic and commensal bacteria can induce autophagy in gut epithelial cells. The induction of autophagy may underlie some of the beneficial clinical effects attributed to a healthy enteric microbiota and probiotic agents.

Ceco-colonic myoelectrical activity was investigated in rats pretreated for 23 weeks by sennosides (10 or 40 mg/kg/day), Na-picosulfate (2.5 or 10 mg/kg/day) or laxative vehicle (control). On the last week of treatment the animals were equipped with Nichrome electrodes on the cecum, the proximal and distal colon. In comparison with controls, sennoside or Na-picosulfate treatment did not induce any significant (p0.05) change in the duration of long spike bursts (LSB) which are associated with phasic contractions. On the last 2 days of treatment the frequency of LSB for 2 h before and 2 h after laxative administration, as well as for 30 min after a 3-gram meal was not significantly (p0.05) different in control and treated animals. Similarly, on the first 2 days, as well as on days 13 and 14, after the end of treatment, no significant (p0.05) difference in the LSB frequency appeared between control and treated animals, in the fasted state or after a 3-gram meal. It is concluded that long-term treatment with sennosides or Na-picosulfate does not induce chronic changes in colonic motility in rats.

Background: Interteukin 1 (IL-1) can influence gut functions by inhibiting gastric acid secretion. This study was performed to investigate the effects of IL-1 on intestinal motility and the mechanisms involved. Methods: The effects of IL- 1β were determined by electromyography in conscious rats with implanted electrodes and a permanent catheter in a lateral brain ventricle. Results: Intracerebroventricular IL-1β (15 ng) administered to fed rats immediately stimulated cecocolonic spike bursts and caused a migrating myoelectric complex pattern after a delay in the small intestine. Tenfold higher doses of peripherally administered IL-1β did not promote similar reactions. The IL-1 antagonist reduced the small intestinal effect of IL-1β and blocked the cecocolonic stimulation. Indomethacin and SC 19220 reduced the small intestinal effects but did not antagonize the increase in cecocolonic contractions. In contrast, α-helical CRF 9–41 blocked the increase of cecocolonic contractions but did not antagonize the IL-1β-induced effects on the small intestine. Conclusion: IL-1β's effects on intestinal motility can be mainly ascribed to a central action. The cecocolonic stimulation may be mediated by brain corticotropin-releasing factor, whereas the small intestinal effects involve a prostaglandin mediation.

Isolated smooth muscle cells from the circular layer of pig and guinea-pig ileum were used to study the effect of prostaglandin E2 (PGE2) and three PGE2 receptor (EP) agonists; iloprost (EP1), butaprost (EP2) and enprostil (EP3). In pig cells, PGE2 and enprostil induced cell contraction (22.1 and 21.5% shortening of cell length, obtained at 10 nM for PGE2 and 1 nM for enprostil, respectively). Iloprost and butaprost had no contractile effect. However, the cholecystokinin octapeptide (CCK-8; 10 nM)-induced contraction was inhibited when cells were preincubated with iloprost or butaprost. In guinea-pig cells, PGE2, butaprost and iloprost induced cell contraction, whereas enprostil had no effect (23.1% for 10 nM PGE2, 22.8% for 1 nM butaprost and 22.6% for 10 nM iloprost). Preincubation with SC19220 (EP1 antagonist) inhibited the PGE2-, butaprost- and iloprost-induced contractions. When the contractile effect of PGE2, butaprost and iloprost was inhibited by addition of SC19220, these agents inhibited the cell contraction induced by CCK-8 (1 nM). Smooth muscle cells from guinea-pig and pig ileum express two PGE2 receptor subtypes that induce opposite effects. EP1 and EP3 receptors mediate cell contraction in guinea-pig and pig, respectively, whereas EP2 receptors mediate cell relaxation in both species.

Recombinant interleukin-1 receptor antagonist protein (rlRAP, 0.5 mg/kg) administered intraperitoneally in guinea pigs one hour before primary and booster parenteral sensitization (1 ml) by cow milk, led to a reduced immunoglobulin E (lgE) production, as displayed by a passive cutaneous anaphylaxis test. rlRAP administered intraperitoneally in sensitized guinea pigs at 0.5 mg/kg 10 min before challenge administration (β-lactoglobulin, 100 mg per os ), also prevents the colonic motor and secretory changes induced by intestinal anaphylaxis. These results suggest the involvement of interleukin-1 in food allergy and evidence a double protective role for rlRAP in food hypersensitivity.

Intracolonic administration of trinitrobenzene sulfonic acid (TNBS) to rats produces chronic colitis associated with an increased release of eicosanoids, platelet-activating factor (PAF), and interleukins.Motor effects of TNBS on proximal colon were evaluated electromyographically in rats. Mediator involvement was investigated using eicosanoids and PAF antagonists.The colonic myoelectrical activity was 59 +/- 17 spike bursts per hour lasting 6.9 +/- 1.3 seconds. Two to eight days after TNBS treatment, spike-burst duration was significantly (P0.05) higher, with a maximal 1.5-4-fold enhancement at day 3. These alterations were significantly (P0.05) reduced by daily treatment with MK-886, a 5-lipoxygenase inhibitor (10 mg/kg, orally), whereas indomethacin (1 mg/kg per day, intramuscularly) was ineffective. At day 3, RP55778, a PAF antagonist (45, 60 mg/kg, intraperitoneally), and rIRAP, an interleukin 1 antagonist (0.3 mg/kg, intraperitoneally) but not KT1-32, a thromboxane A2 antagonist (30, 60 mg/kg orally), nor SKF104,353, a leukotriene D4 antagonist (2, 4 mg/kg, orally), significantly (P0.05) reduced the TNB-induced motor effects.TNBS-induced colitis in rats involves a delayed long-lasting dysmotility involving PAF, interleukin 1, and some leukotrienes but not leukotriene D4, thromboxane A2, or other cyclo-oxygenase products.

In an attempt to establish the role of F8Famide in opioid activity modulation, we examined the effects of intracerebroventricular administration of the F8Famide analogs (1DME)Y8Fa and (3D)Y8Fa on intestinal transit in mice. (1DME)Y8Fa (0.88 to 22 nmol) inhibited intestinal transit as did F8Famide and morphine. An IP injection of naloxone (2 mg/kg) decreased the morphine effect but had no effect on the response to (1DME)Y8Fa. In contrast, a subthreshold dose of morphine (0.22 nmol) inhibited the response to (1DME)Y8Fa. (3D)Y8Fa delayed intestinal transit only at large dose (22 nmol) but decreased (1DME)Y8Fa and morphine effects at lower ineffective doses. Our findings demonstrate that although F8Famide and morphine could induce the same pharmacological effect, F8Famide receptor activity was modulated by a low-level stimulation of opioid receptors. Furthermore, (3D)Y8Fa should be a useful probe to elucidate neuronal mechanisms controlled by opioids.

The role of 5-HT1, 5-HT2 and 5-HT3 receptors in the genesis of colonic motor alterations induced by emotional stress was evaluated in rats equipped with implanted nickel/chrome electrodes on the proximal colon and a catheter into the lateral ventricle of the brain. In control rats the frequency of colonic spike bursts increased from 7.6 ± 1.3 to 16.8 ± 1.3 per 10 min when the rats were placed in a test cage in which they had previously received electric footshocks. I.p. injection of methysergide (0.1 mg/kg) reduced by 54% the emotional stress-induced increase of colonic spike burst frequency, while a higher dosage (1 mg/kg) of methysergide had no effect. The i.p. injection of ketanserin (a 5-HT2 receptor antagonist, 0.1 and 1 mg/kg) or granisetron (a 5-HT3 receptor antagonist, 0.1 and 1 mg/kg) had no effect on emotional stress-induced colonic hyperkinesia. The i.p. injection of the 5-HT1A receptor agonists, buspirone (1 mg/kg) or 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) (0.05 and 0.1 mg/kg) or benzodiazepine (clonazepam, 1 mg/kg) significantly reduced or suppressed the emotional stress-induced increase of colonic spike bursts. Injected i.c.v., buspirone, but not 8-OH-DPAT, also reduced the emotional stress-induced hyperkinesia. Pretreatment with devazepide receptor (1 μg/kg) antagonized the inhibitory effects of buspirone and 8-OH-DPAT injected i.p. on emotional stress-induced colonic hyperkinesia but did not alter the effects of clonazepam (1 mg/kg). These results suggest that, in rats, (i) 5-HT1 but not 5-HT2 and 5-HT3 receptors are involved in the mediation of emotional stress-induced stimulation of colonic motility and (ii) 5-HT1A receptor agonists inhibit stress-induced colonic disturbances by activating central cholecystokinin neurons through an afferent pathway from a peripheral site of action.

The central and peripheral effects of clonazepam (central benzodiazepine receptor agonist) on intestinal myoelectrical activity and the origin of the effects were evaluated in conscious rats, chronically fitted with Nichrome electrodes implanted on the jejunum and with an intracerebroventricular (i.c.v.) cannula. Administered intraperitoneally (i.p.) in 12-h fasted rats, clonazepam (0.05 to 0.5 mg/kg) dose dependently disrupted jejunal cyclic migrating myoelectric complexes, characterizing the fasted state, which were replaced by a permanent irregular spiking activity, lasting 259 +/- 37 min for clonazepam at the dose of 0.5 mg/kg. This disruption of migrating myoelectric complexes occurred after a delay which increased with increasing clonazepam doses. In contrast, injected i.c.v. at doses from 1 microgram/kg to 1 mg/kg, clonazepam did not alter the migrating myoelectric complexes pattern of the small intestine. Injected i.p., flumazenil (central benzodiazepine receptor antagonist) (1 mg/kg) but not PK 11-195 (peripheral benzodiazepine receptor antagonist) (5 mg/kg) suppressed the effects of i.p. clonazepam (0.1 mg/kg). Administered i.c.v., 10 min prior to clonazepam (0.1 mg/kg i.p.), devazepide (CCKA receptor antagonist) at a dose as low as 10 ng/kg reduced the migrating myoelectric complex disruption induced by clonazepam. L365-260 (CCKB receptor antagonist) administered i.c.v reduced the migrating myoelectric complex disruption at 10-fold higher doses and loxiglumide (CCKA receptor antagonist) injected i.c.v, at 100-fold higher doses. When administered i.p. neither devazepide nor L365-260 affected the duration of migrating myoelectric complex disruption induced by clonazepam (0.1 mg/kg i.p.) or its delay of occurrence at doses lower than 0.1 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduction: Lactobacillus plantarum 299v is a probiotic strain indicated to treat IBS patients. No other trial so far has tested the symptomatic effect of this strain in such a large population sample. Aims & methods: The aim of this multicenter double blind randomized placebocontrolled trial was to determine the efficacy of L.plantarum 299v in a powerful trial. Patients from 19 to 70 years old and suffering from chronic IBS according to Rome III criteria were recruited in 3 Indian centers. Treatment duration was 4 weeks long with per day one cap of placebo or one cap of 1010 cfu of L.plantarum 299v. Both frequency and intensity of abdominal pain, bloating, feeling of incomplete rectal evacuation and the number of stools were assessed by a VAS. The results are expressed as the percentage of improvement between baseline and the end of the treatment period. Overall patients' assessment was expressed on a 4-point rated scale. Significance analyses were made by using Chi-square or ANOVAR tests. Results: The ITT population comprised 200 patients and only 10 patients dropped out (PP population: 190). Neither the demographic characteristics nor the diet were different between the two treatment arms (see table 1). At the end of treatment, L.plantarum 299v improved significantly the symptoms both compared to baseline and to placebo (see table 2). Overall patients' assessment was significantly better in the L.plantarum 299v group compared to the placebo group (see table 2). Pulse and respiratory rates, blood pressure and internal temperature remained normal throughout the study without any significant changes in both arms. No side-effect was reported in both groups. Conclusion: A 4 weeks treatment with Lactobacillus plantarum 299v has shown to be very effective for the relief of symptoms, particularly abdominal pain and bloating in IBS patients fulfilling the Rome III criteria. Table 2: Clinical Evaluation and Patients' assessment

10+3 sec vs 250+20 sec; P 0.05). Microdialysis of the glutamate NMDA receptor antagonist AP5 (100 μM) into the ACC before but not after conditioning (CPD), blocked the acquisition of CPA learning (-2+0.5 sec vs 200+5 sec sham control; P

Background: Studies have suggested obesity as a risk factor for colorectal adenoma recurrence. It is unknown if weight change affects the risk of adenoma recurrence. Aim: We sought to examine whether weight change over a four year period is associated with subsequent colorectal adenoma recurrence. Methods: A total of 1,826 participants with a history of colorectal adenoma had body weight measured at baseline and 4 years later in the Polyp Prevention Trial. Adenoma recurrence was determined by end of trial colonoscopy. We used multinomial regression models to estimate adjusted relative risk ratios (RRR) and 95% confidence intervals (CI) to evaluate body mass index (BMI), weight change over 4 years and the risk of incident non-advanced and advanced adenoma recurrence. Results: A total of 605 (33.1%) participants had non-advanced adenoma recurrence while 118 (6.5%) had advanced adenoma recurrence. Although relative risks in this data set did not quite reach statistical significance, the analytic results suggest that baseline adiposity, especially BMI ≥ 30 kg/m2, is positively associated with adenoma recurrence. When compared with participants with BMI < 25 kg/m2 (n = 466) at baseline, there was an elevated risk of nonadvanced adenoma and advanced adenoma recurrence among participants with BMI = 25 29 kg/m2 (n = 868) (RRR = 1.23; 95%CI: 0.95-1.59 and RRR = 1.02; 95%CI: 0.61-1.68, respectively) and among those with BMI ≥ 30 kg/m2 (n = 492) (RRR = 1.27; 95%CI: 0.951.70 and RRR = 1.63; 95%CI: 0.96-2.78, respectively). When compared with those with relatively stable weight (less than 5 pound weight change) over the 4-year trial, weight gain or loss was not associated with adenoma recurrence (Table); this was consistent regardless of the baseline BMI. Conclusions: We observed a suggestive positive association between baseline BMI and adenoma recurrence; however, weight loss or gain over 4 years does not affect adenoma recurrence irrespective of baseline weight. Adjusted for age, sex, non-steroidal anti-inflammatory drug use, smoking and family history of colorectal cancer

1. In order to determine the intracellular mechanisms by which galanin induces contraction of isolated smooth muscle cells from pig ileum, we examined the effects of external Ca2+, relaxing agents, pertussis toxin and forskolin on the galanin-induced contraction and compared these effects to those observed on the cholecystokinin derivative CCK8-induced contraction. 2. Galanin induced a concentration-dependent cell contraction. The maximal contraction (24.5 +/- 2.1% of the length of resting cells) was observed at 1 nM of galanin. When cells were incubated in the simultaneous presence of concentrations of galanin (10 fM) and CCK8 (1 pM) which were ineffective alone, or galanin (10 fM) and acetylcholine (100 pM), a synergistic action was observed corresponding to a submaximal contraction. 3. Incubation of cells in Ca(2+)-free medium caused a significant decrease in galanin- but not in CCK-induced contraction. Nifedipine, a Ca2+ channel blocker, provoked a concentration-dependent inhibition of galanin-induced contraction while it had no effect on the contraction induced by CCK8. 4. Vasoactive intestinal polypeptide (VIP) and isoprenaline, known to induce cell relaxation through an increase in intracellular cAMP level, inhibited CCK-induced cell contraction at concentrations ranging from 1 pM to 1 microM but failed to inhibit cell contraction induced by galanin. 5. When cells were pre-incubated for 3 h in the presence of 200 ng/ml of pertussis toxin, the contraction induced by galanin was abolished while the CCK-induced contraction remained unchanged. On the contrary, 10 microM forskolin abolished the contraction induced by 10 nM CCK but had no effect on galanin-induced contraction. 6. These results indicate that galanin induces a concentration-dependent contraction of pig ileum smooth muscle by a direct myogenic effect. This effect of galanin involves the activation of a pertussis toxin-sensitive G protein, which results in an influx of Ca2+ into the cell. This intracellular pathway is insensitive to the relaxing effect of cAMP.

Using an intraluminal probe with six pairs of annular electrodes, the myoelectric activity of the proximal jejunum was recorded during 48-hr sessions in 16 healthy volunteers receiving evening and noon meals (1000 kcal) and breakfast (400 kcal). In 10 subjects receiving no drug, the characteristics of the migrating motor complexes (period, duration of each phase, velocity of propagation of phase 3, duration of the postprandial disruption) varied markedly between subjects but were relatively constant from the first to the second day of recording. Single spike bursts propagated at a rate of 2–5 cm/sec, clusters of 3–10 spike bursts propagated at a rate of 0.5–1 cm/sec, and similar clusters recurring repetitively each 1.5–2 min were observed after the meals and very rarely in the fasted state during phase 2 of nocturnal migrating motor complexes. In six subjects, oral administration of codeine (50 mg) 1 hr before a meal induced migrating motor complexes in the postprandial state, with characteristics similar to that observed in the fasted state except a longer duration of phase 2. Single spike bursts and isolated and repetitive clusters of spike bursts were observed during phase 2 of the codeine-induced migrating motor complexes and after meals preceded by placebo, but very rarely during the phase 2 of nocturnal (fasted state) migrating motor complexes. It is concluded that the patterns of jejunal contractions consisting of propagated single spike bursts and isolated or repetitive spike bursts characterize the postprandial state in healthy humans and are dependent upon digesta flow.

An ultrasonographic measurement of the gallbladder volume based on an ellipsoid approximation of the gallbladder shape was validated in vivo in the dog. The mean difference between the ultrasonographically determined and the true gallbladder volumes amounted to 2.0 ± 1.6 cm 3 ( X ± SD ) , whereas the regression line computed for the ultrasonographically measured versus true gallbladder volumes was y = 0.892x + 3.0, r = 0.955, p ⪡ 0.001. The ultrasonographic method enabled a noninvasive, repeatitive measurement of either a meal- or caerulein-induced gallbladder emptying in the dog.

Effect of galanin and CCK8 were studied on isolated smooth muscle cells obtained from pig, guinea-pig, rat, rabbit and dog ileum circular muscle layer. Galanin as well as CCK8 induced a concentration-dependent contraction of pig, rat, rabbit and guinea-pig ileum smooth muscle cells. Maximal contraction ranged between 23.7 ± 1.9% and 26.1 ± 3.1% decrease in cell length from control in the presence of both peptides. This maximal contraction was obtained at 1 nM galanin in pig, rat, rabbit, 1 nM CCK8 in rat, rabbit, guinea-pig, at 10 nM galanin in guinea-pig and 10 nM CCK8 in pig. Concentrations of galanin inducing a half maximal contraction (EC50) ranged between 8 pM and 80 pM in these species. In dog, CCK8 induced a concentration-dependent contraction of ileum smooth muscle cells, with a maximal contraction (24.5 ± 2.3%) at 1nM and an EC50 of 50 pM while.galanin inhibited cell contraction induced by CCK8. The CCK-induced contraction was abolished at 10 nM galanin and 10 nM VIP. Concentrations of galanin and VIP inducing a half-maximal relaxation of contracted cells were 2 pM and 3 pM respectively. It is concluded that galanin may induce cell contraction of pig, guinea-pig, rat and rabbit ileum circular muscle layer and cell relaxation of dog ileum by a direct myogenic effect.

The effects of orally administered Tyr-MIF-1, an agonist of an endogenous antiopiate system, were examined on gastric emptying in mice and gastrointestinal myoelectric activity in rats. Tyr-MIF-1 (5 mg/kg in mice, 20 mg/kg in rats) accelerated gastric emptying of a methylcellulose test meal, increased the frequency of antral spike bursts, and disrupted intestinal migrating myoelectric complexes. These effects were reproduced by a subcutaneous administration of Tyr-MIF-1 at the same dosage. They were blocked by naloxone (1 mg/kg) but not by the kappa receptor subtype antagonist MR 2266 (1 mg/kg). The GABAA antagonist bicuculline (0.5 mg/kg), but not the GABAB antagonist 2-hydroxysaclofen (4 mg/kg), also antagonized the effects of Tyr-MIF-1. These data demonstrate that oral Tyr-MIF-1 stimulates gastric emptying and gastrointestinal motility through a systemic or central action that involves opioid and GABA systems.

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The effects of two degranulators of mast cells and intestinal anaphylaxis on jejunal myoelectric activity were compared in rats fasted for 15 hours. Attempts to antagonize the motility changes were performed using antagonists of histamine and serotonin and a cyclooxygenase and lipoxygenase inhibitor. Hooded Lister rats were chronically fitted with electrodes implanted in the jejunal wall. A group of rats was sensitized to egg albumin and challenged 14 days later by intraduodenal infusion of antigen. Sensitized animals had serum titers greater than or equal to 1:64. The other group was administered with mast cells degranulators. Both 48/80 (1 mg/kg), a degranulator of connective mast cells, and bromolasalocid (2 mg/kg), acting on connective and mucosal mast cells, induced a phase of total spiking inhibition followed by a progressive irregular spiking activity until the recovery of migrating myoelectric complex pattern (about 3 hours after injection). In contrast, antigen challenge disrupted the migrating myoelectric complex pattern, which was replaced by a peculiar pattern characterized by propagated spike burst, lasting 98 +/- 11.3 minutes. Chlorpheniramine (1 mg/kg) antagonized only the inhibitory phase induced by degranulators and was ineffective on the intestinal anaphylaxis-induced motor changes. Methysergide (1 mg/kg) and indomethacin (5 mg/kg) significantly reduced the degranulator effects as well as the anaphylaxis-induced alterations of intestinal motility. It is concluded that anaphylaxis-induced motor disturbances are relevant to mucosal mast cell degranulation involving 5-hydroxytryptamine and arachidonic acid derivative products, whereas histamine release appears to be a minor component.

The effects of central and peripheral administration of a prostaglandin E2 analogue (enprostil) on colonic transit time, faecal dry matter and colonic myoelectrical activity, were examined in rats chronically fitted with a cannula in a lateral ventricle of the brain and a catheter inserted in the lumen of the proximal colon, or nichrome electrodes implanted on the proximal colon. In control studies, the mean retention time (MRT) of a marker ([51Cr] sodium chromate) administered into the proximal colon and determined in the faeces collected at hourly intervals was 7.3 ± 1.7 h, the frequency of colonic long spike bursts was 59.1 ± 9.1/h and faecal dry matter (DM) 65.4 ± 3.4%. Enprostil administered orally (350 μg/kg) decreased colonic MRT (4.9 ± 0.6 h) and faecal DM (50.1 ± 3.2%) and increased by 68% the colonic myoelectric index. Administered intracerebroventricularly enprostil (12 μg/kg) also accelerated colonic transit (MRT = 5.1 ± 1.6 h) and decreased faecal DM (48.7 ± 3.4%) but did not modify colonic spiking activity. Intraperitonal administration of the PG receptor antagonist SC-19220 (1 mg/kg) did not affect the effects of enprostil administered centrally on MRT and DM but blocked the action of oral enprostil on colonic transit time, DM and colonic spiking activity. Centrally administered, SC-19220 (100 μg/kg) antagonized the effects of oral enprostil on colonic transit and faecal DM but not on colonic spiking activity. It is concluded that in rats the enprostil-induced acceleration of colonic transit involves a peripheral action on colonic motility and a secretory component which is centrally mediated. Moreover these results show that colonic hypersecretion, but not hypermotility, can alone accelerate colonic transit.

The effects of colonic inflammation induced by trinitrobenzene sulfonic acid and influence of previous treatment with specific antagonists of inflammatory mediators (platelet-activating factor, leukotrienes, prostaglandins, and thromboxanes) on colonic transit were examined in conscious rats which were permanently fitted with an intracolonic catheter inserted into the proximal colon. Colonic inflammation was induced by intracolonic administration of trinitrobenzene acid (80 mg/kg) in 50% ethanol. Colonic transit time was evaluated by intracolonic administration of a radiolabeled marker [( 51Cr]sodium chromate) and collection of the feces per hour on a conveyor belt. Excretion of the marker was then plotted vs. time, permitting calculations of the times elapsed to recover 25%, 50%, and 75% of the marker injected (T25, T50, and T75, respectively). In control (saline) animals, excretion of the marker described a regular sigmoid curve with 50% of the marker recovered at 6.92 +/- 0.40 hours after intracolonic administration (T25 = 6.4 +/- 0.43 hours; T75 = 7.49 +/- 0.39 hours). Ethanol (vehicle), 50%, did not modify the profile of marker recovery. On the contrary, single intracolonic administration of trinitrobenzene sulfonic acid/ethanol induced a biphasic response consisting of an early pool of radiolabeled feces (T25 = 4.03 +/- 0.55 hours) with a delayed total one (T50 = 11.74 +/- 0.83 hours; T75 = 13.70 +/- 0.49 hours). Antagonists of the leukotriene pathway, i.e., MK = 886, a lipoxygenase inhibitor, and SKF 104,353 and SR 2640, two different leukotriene D4 receptor antagonists, blocked the effects of trinitrobenzene sulfonic acid on colonic transit time and restored a control profile of radiolabeled marker excretion. In contrast, indomethacin, a cyclooxygenase inhibitor, and SC 19220, a specific prostaglandin E2 receptor antagonist, were inefficient in blocking the effects of trinitrobenzene sulfonic acid on colonic transit time. Specific thromboxane A2 receptor antagonists, KT1-32 and GR 32191B, did not show any improvement in colonic transit after trinitrobenzene sulfonic acid administration. Previous injection of the specific platelet-activating factor receptor antagonists, BN 52021 or BN 50730, was also unable to restore a normal marker excretion profile after administration of trinitrobenzene sulfonic acid. It is concluded that the alterations of colonic transit immediately observed after intracolonic trinitrobenzene sulfonic acid administration are mediated through the release of leukotriene D4. In contrast, platelet-activating factor, prostaglandins, and thromboxanes are not involved in the mediation of these transit disturbances.

This work was performed to compare withdrawal symptoms induced by the administration of the central vs. peripheral benzodiazepine antagonists in rats treated chronically with diazepam (15 mg/kg, SC) for 8 days. Withdrawal was expressed as motor, autonomic, and behavioral signs. Significant withdrawal occurred after the administration of both flumazenil (15 and 20 mg/kg, IP) and PK11195 (5 and 10 mg/kg, IP). With these doses, PK11195 induced diarrhea and decreased motor activity more than did flumazenil. These preliminary results suggest that peripheral benzodiazepine receptors are involved in the withdrawal syndrome in diazepam-dependent rats.

Early epithelial restitution is an important repair modality in the gut mucosa and occurs as a consequence of epithelial cell migration. Our previous studies have shown the canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs) and regulates early mucosal restitution. Increased TRPC1 channel activity increases store-operated Ca2+ entry (SOCE) and enhances IEC migration during restitution. However, the exact upstream signals initiating TRPC1 activation after mucosal injury remain elusive. Recently, stromal interaction molecule 1 (STIM1) has been identified as a store Ca2+ sensor and can rapidly translocate to the plasma membrane (PM) where it interacts with and modulates Ca2+ channels. We hypothesized that STIM1 is crucial for stimulation of IEC migration after wounding by activating TRPC1 channel activity.Methods: Studies were conducted in IEC-Cdx2L1 cells, which represent differentiated IECs. STIM1 subcellular distribution was analyzed by surface biotinylation assays and immunohistochemical staining, while its functions were investigated by specific siRNA (siSTIM1) and ectopic overexpression of constitutively active STIM1 EF-hand mutants. [Ca]cyt was measured by fluorescence digital imaging analysis. Cell migration was measured in a model that mimics cell division-independent epithelial restitution. Results: IECs highly expressed STIM1 in cultured cells and in the intestinal mucosa of the rat. STIM1 translocation to the PM increased significantly within 10 min after wounding, peaked between 30 to 60 min, and then gradually returned to normal. Maximum increases in levels of STIM1 at PM were ~six times the prewounding control, which was associated with a dramatic increase in SOCE. There were no changes in total STIM1 protein levels after wounding. STIM1 directly interacted with TRPC1 in the PM as measured by immunoprecipitation assays. Ectopic expression of constitutively active STIM1 EF-hand mutants increased SOCE (by ~60%) and also stimulated IECmigration (by ~35%) after wounding. In contrast, STIM1 silencing by siSTIM1 decreased SOCE, inhibited IEC migration, and delayed epithelial restitution in cells overexpressing TRPC1. Levels of Ca2+ influx due to SOCE were decreased by ~70% in STIM1-silenced populations, whereas cell migration was inhibited by ~45%. Conclusions: These findings indicate that 1) wounding increases STIM1 translocation to the PM in IECs and 2) STIM1 at the PM interacts with and activates TRPC1, thus stimulating epithelial restitution as a result of increase in SOCE.

Introduction: Patients with IBD show higher thresholds to slow ramp distension of the rectum, as opposed to patients with a diarrhea-predominant IBS (IBS-D). Recently we have found elevated serine protease (SerP) activities in both IBS-D and ulcerative colitis (UC) fecal materials. In mice, intracolonic (IC) infusion of fecal IBS-D supernatants evokes a proteinase-activated receptor (PAR)-2 mediated colonic hypersensitivity to distension. Objectives: Our aims were to evaluate the influence of fecal supernatant from UC patients on visceral sensitivity in mice and to test the involvement of PAR-4, and its activator cathepsin G (cat-G) as possible mediators in this nociceptive response. Methods: Fecal samples from UC patients and healthy subjects were dissolved in saline, centrifuged and filtered. Under anaesthesia electrodes were inserted in the abdominal muscle to record abdominal cramps in C57/BL6 male mice. Four days after surgery mice were IC infused with 0.3 mL fecal supernatant from UC patients or controls, and colorectal distensions (CRD) were performed using a balloon inflated from 0 to 0.12 mL each steps lasting 10 sec with 5 min intervals. To evaluate the role of PAR-4, micewere treatedwith a PAR-4 antagonist (P4pal-10, pepducin, 3x0.25 mg/kg) IP before and during UC supernatant infusion. The participation of cat-G was assessed by incubating the UC supernatant with a mixture of antiproteases (aprotinin and SBTI, 0.22 and 1.70 mg/mL respectively) or with a cat-G inhibitor (0.057 mg/mL). The effect of PAR-4 activation or cat-G was tested by IC infusion of 100 ug PAR-4 agonist (PAR4AP, 0.667mg/mL) or 0.025 UN cat-G (0.167UN/mL). Results: IC infusion of UC supernatants decreased the intensity of abdominal EMG response by 71%, 49% and 35% for 0.04, 0.06 and 0.08 mL of CRD (p

Introduction La prise en charge du syndrome de l’intestin irritable (SII) represente une part importante de l’activite du gastroenterologue. La place des traitements alternatifs y est mal connue. L’objectif de cette enquete nationale etait d’evaluer pour la premiere fois le recours a ces therapeutiques et le niveau de satisfaction des patients. Patients et Methodes Les patients inclus etaient adultes et consultaient un gastroenterologue pour un SII. Un questionnaire medical precisait la symptomatologie et les traitements medicamenteux ou alternatifs. Un auto-questionnaire patient anonyme portait sur les professionnels de sante consultes, l’information recue et le niveau de satisfaction vis-a-vis des traitements. Parmi les 790 gastroenterologues sollicites, 644 (81,5 %) ont participe a cette enquete menee de mars a septembre 2008. 2 933 paires de questionnaires medecin-patient etaient analysables. Resultats Les patients etaient en majorite des femmes (72 %), etaient âges de 52 ± 16 ans et souffraient d’un SII depuis 10 ± 11 ans. Il s’agissait d’un nouveau patient pour le gastroenterologue dans 37 % des cas. 82 % des patients estimaient avoir recu l’information souhaitee sur leur SII. Parmi les symptomes les plus frequemment observes on notait : douleur : 91 %, ballonnements : 62 %, constipation : 37 %, epigastralgies : 22 %). Sur une echelle visuelle analogique allant de 0 a 10 le niveau de satisfaction generale des patients sur leur prise en charge etait de 5,1 ± 2,5 et 28 % avaient un niveau de satisfaction ≤ 3. Vis-a-vis du traitement medicamenteux traditionnel, le niveau de satisfaction etait de 4,9 ± 2,4 et 31 % des patients avaient un niveau de satisfaction ≤ 3. 20 % des patients avaient eu recours au cours des 12 derniers mois a un ou plusieurs traitements alternatifs (13 % selon les medecins, p Conclusion Au cours du SII, plus de 80 % des patients considerent leur niveau d’information comme satisfaisant. Les gastroenterologues sous-estiment le recours de leurs patients a des traitements alternatifs. Ce recours est lie a l’anciennete du SII, l’alternance diarrhee-constipation, les ballonnements et l’anxiete-depression. Ces diverses therapeutiques alternatives ne donnent pas un niveau de satisfaction superieur aux traitements traditionnels.

Introduction L’influence des probiotiques sur la reponse de l’axe HPA au stress est tres peu documentee. Il a ete montre qu’un traitement probiotique supprime l’hypercorticosteronemie induite par un stress neonatal chez le rat [1]. Toutefois, l’effet d’un traitement probiotique sur la reponse immediate de l’axe HPA en reponse a un stress aigu n’a pas ete evalue. Ainsi, l’objectif de cette etude etait d’evaluer l’effet d’un traitement par Lactobacillus farciminis (L.f) sur les modifications de la reponse de l’axe HPA induites par un stress aigu par determination (i) des taux plasmatiques de corticosterone (CS) et de l’hormone corticotrope (ACTH) (ii) de l’activation neuronale et de l’expression du facteur de liberation de corticotrophine (CRF) au niveau du noyau paraventriculaire de l’hypothalamus (PVN). Materiels et Methodes Dans une premiere serie d’experimentations, 14 groupes de 5 rats femelles Wistar recevant par voie orale L.f (1011 UFC/jour) pendant 15 jours (groupes 1-7) ou du NaCl 0,9 % (groupes 8-14) ont ete utilises, afin d’etablir une mesure cinetique des taux plasmatiques de CS et d’ACTH. Tous les animaux ont ete soumis a un stress de contrainte excepte ceux des groupes 1 et 8 (stress fictif, conditions basales). Apres 15, 30, 45, 60, 90 et 120 minutes de stress, un prelevement sanguin au niveau de l’aorte abdominale a ete realize pour la mesure des taux de CS et ACTH a l’aide d’un kit de dosage Linconplex. Dans une deuxieme serie, 4 groupes de 4 rats femelles Wistar traites par voie orale par L.f (1011 UFC/ jour) pendant 15 jours (groupes 1-2) ou du NaCl 0,9 % (groupes 3-4) ont ete soumis a une session de stress (groupes 1-3) ou de stress fictif (groupes 2-4). A la fin de la session de stress le cerveau a ete preleve et un co-marquage par immunofluorescence de la proteine c-Fos (marqueur d’activite neuronale) et du CRF a ete realise sur des coupes de la region PVN. Resultats Une augmentation (P Conclusion Cette etude montre qu’un traitement de 15 jours par L farciminis reduit la corticosteronemie en conditions basales et normalise l’activation neuroendocrine en reponse a un stress aigu chez le rat.

Introduction Contrairement aux patients presentant un SII a predominance diarrheique (SII-D), les patients souffrant de RCH presentent une augmentation du seuil de sensibilite en reponse a la distension rectale [1,2]. Recemment, nous avons decrits des taux d’activite proteasique eleves dans les feces de patients SII-D et de patients RCH. Chez la souris, la perfusion intracolique (IC) de surnageants de patients SII-D provoque une hypersensibilite viscerale via l’activation des recepteurs aux proteases (PAR) -2. Nos objectifs etaient d’evaluer l’impact d’une perfusion IC de surnageants de patients RCH sur la sensibilite viscerale en reponse a la distension colorectale (DCR) chez la souris et de tester l’implication des PAR-4 et de son activateur endogene, la cathepsine-G dans cette reponse. Materiels et Methodes Des echantillons de feces de sujets controles, de patients SII-D et de RCH ont ete homogeneises dans du NaCl 0,9 %, centrifuges et filtres. L’activite myoelectrique des muscles abdominaux a ete enregistree chez des souris C57/BL6 pour apprecier la reponse douloureuse a des DCR, 60 minutes apres perfusion IC (300 μL) de surnageants de patients SII-D, RCH ou de sujets controles. Le role des PAR-4 a ete evalue apres traitement des animaux a un antagoniste des PAR-4 (pepducine, 3 × 0,25 mg/ kg) administre par voie intra-peritoneale (IP) avant la perfusion IC des surnageants de patients RCH. La participation des proteases et de la cathepsine-G dans la reponse a la DCR ont ete evalues par pre-incubation des surnageants de patients RCH avec un melange d’antiproteases (aprotinine et SBTI) ou d’un inhibiteur de cathepsine-G. L’effet de l’activation des PAR-4 ou de la cathepsine-G sur la douleur viscerale a ete evalue apres perfusion IC d’un agoniste PAR-4 (0,667 mg/mL) ou de cathepsine-G (0,167 UN/mL). Resultats A l’inverse des surnageants SII-D, la perfusion IC de surnageant de patients RCH diminuait significativement l’intensite des contractions musculaires abdominales en reponse a de faible volumes de DCR par rapport a la perfusion IC de surnageants sujets controles (71 %, 49 % et 35 % respectivement pour 0,04, 0,06, et 0,08 mL). L’antagonisme des recepteurs PAR-4 a non seulement supprime cette hyposensibilite mais a provoque une hypersensibilite colique similaire a celle mesuree apres perfusion IC de surnageant (SII-D). De la meme facon une pre-incubation des surnageants RCH avec un inhibiteur de cathepsine-G se traduisait par une hypersensibilite viscerale. Au contraire, une inhibition totale de l’activite proteasique des surnageants RCH par un melange d’antiproteases a bloque toute reponse douloureuse a la DCR. Enfin, la perfusion IC de l’agoniste PAR-4 ou de la cathepsine-G ont diminue de facon significative la sensibilite viscerale en reponse a la DCR. Conclusion Malgre des taux eleves d’activite proteasique similaires a ceux mesures dans les feces de patients SII-D, les surnageants fecaux de patients RCH reduisent la sensibilite colorectale chez la souris. Cette hyposensibilite viscerale implique l’activation des PAR-4 par la cathepsine-G endoluminale.

The effects of IV administration of Escherichia coli endotoxin on intestinal myoelectric activity was investigated in conscious fasted rats chronically implanted with nichrome electrodes in the duodenojejunum. These effects were compared with those of platelet-activating factor and were evaluated in animals pretreated with a specific platelet-activating factor antagonist, BN 52021, indomethacin, a selective prostaglandin E2 antagonist, SC 19220, and several free radical scavengers. Intravenous administration of endotoxin (E. coli S.O111:B4) at a dose of 50 micrograms/kg suppressed the migrating myoelectric complexes, which were replaced by continuous rhythmic clusters of rapidly propagated spike bursts for 114.7 +/- 19.9 minutes. Intraperitoneal platelet-activating factor (25 micrograms/kg) also inhibited the migrating myoelectric complex pattern for 146.1 +/- 24.1 minutes. Previous IV administration of BN 52021 (50 mg/kg-1) abolished the motor alterations induced by platelet-activating factor and significantly reduced to 43.1 +/- 12.2 minutes those induced by endotoxin (P less than 0.01). Indomethacin (10 mg/kg IP), injected before endotoxin or platelet-activating factor, also significantly reduced the duration of migrating myoelectric complex inhibition to 45.6 +/- 7.8 and 47.7 +/- 8.3 minutes, respectively (P less than 0.01). SC 19220 significantly reduced the effects of platelet-activating factor from 151.8 +/- 26.4 to 67.4 +/- 14.7 min (P less than 0.01). Superoxide dismutase (15,000 U/kg IV) injected before either endotoxin or platelet-activating factor shortened the migrating myoelectric complex inhibition to 45.7 +/- 9.9 and 72.9 +/- 10.4 minutes, respectively (P less than 0.01). Allopurinol and dimethylsulfoxide administered orally at 50 mg/kg 1 hour before endotoxin reduced the migrating myoelectric complex inhibition to 42.5 +/- 6.5 and 38.2 +/- 6.4 minutes, respectively (P less than 0.01). They also reduced platelet-activating factor-induced intestinal myoelectric alterations to 68.5 +/- 10.6 and 31.7 +/- 6.1 minutes, respectively (P less than 0.01). It is concluded that endogenous release of platelet-activating factor is partly responsible for the intestinal motor alterations induced by endotoxin, these effects being also mediated through the release of prostaglandins and free radicals. However, prostaglandins, as well as free radicals, appear to be partly involved in the platelet-activating factor-induced action of E. coli endotoxin on intestinal motility.

The effect of a mental stress model corresponding to conditioned fear on cecocolonic motility was evaluated electromyographically in intact and hypophysectomized rats equipped with electrodes implanted in the cecum and proximal colon over a long period and a small polyethylene catheter inserted into the right lateral ventricle of the brain. Intact fasted and fed rats showed an increase of 82.3% and 67.2%, respectively, in colonic spike-burst frequency when placed for 30 minutes in a box in which they had previously received electrical shocks in their feet. Intracerebroventricular administration of corticotropin-releasing factor (0.5 micrograms/kg) mimicked the effects of mental stress and increased cecocolonic spike-burst frequency by 75.8%. The specific corticotropin-releasing factor receptor antagonist alpha-helical CRF9-41 given intracerebroventricularly (5 micrograms/kg) prevented both the effects of mental stress and corticotropin-releasing factor (0.5 micrograms/kg intracerebroventricularly) on colonic spike-burst frequency. In contrast, diazepam (0.5 mg/kg IM) suppressed colonic hypermotility induced by mental stress but not that resulting from intracerebroventricular injection of corticotropin-releasing factor (0.5 micrograms/kg). Increased colonic spike-burst frequency induced either by stress or by central administration of corticotropin-releasing factor was not prevented by hypophysectomy. It was concluded that mental stress increases the frequency of cecocolonic spike-burst activity and that these effects are related to the central release of corticotropin-releasing factor because they are blocked by a corticotropin-releasing factor antagonist and reproduced by intracerebroventricular administration of corticotropin-releasing factor. Moreover, mental stress-induced colonic motor alterations are mediated by the autonomic nervous system rather than by the hypothalamopituitary axis because they are not abolished by hypophysectomy.

The effects of galanin and its interaction with cholecystokinin and acetylcholine on smooth muscle cells were studied in vitro on isolated cells obtained from pig ileum circular muscle layer. Galanin induced a concentration-dependent cell contraction with a maximal contraction (24.5% decrease in cell length from control) obtained at 1 nM. The concentration of galanin inducing a half-maximal contraction was 3 pM. Tetrodotoxin (10 microM) failed to inhibit cell contraction induced by galanin (1 nM), pentagastrin (10 nM) and acetylcholine (1 microM). Atropine abolished the contraction induced by acetylcholine (1 microM), but had no effect on galanin- and pentagastrin-induced contraction. L 364,718 inhibited the contraction induced by CCK8 but not the galanin-induced contraction. At the uneffective concentration of 10 fM, galanin had a synergistic effect with an uneffective concentration of CCK8 (1 pM). These results suggest that (i) galanin contracts smooth muscle cells from pig ileum by acting on a specific receptor; (ii) galanin and either CCK or acetylcholine may act in a synergistic way to induce cell contraction.

The effects of hypothalamic microinfusions of cholecystokinin octapeptide and its antagonist L364,718 on cecocolonic myoelectrical activity were evaluated by electromyography in fasted and fed rats. The rats were chronically fitted with electrodes implanted on the cecum and proximal colon and cannulas placed bilaterally in either the ventromedial or lateral hypothalamus. In fasted rats, microinfusion of cholecystokinin octapeptide (10 ng/kg) into the ventromedial hypothalamus increased the spike-burst frequency of the cecum and the colon by 45.6% and 43.7%, respectively, during the 30-minute period after treatment. The injection of cholecystokinin octapeptide (10 ng/kg) into the lateral hypothalamus had no effect on either cecal or colonic motility. Feeding increased the frequency of cecal and colonic spike bursts by 52.1% and 50.1% for 30 minutes postprandially. When infused bilaterally into the ventromedial hypothalamus 10 minutes before feeding, L364,718 (1 or 5 micrograms/kg) abolished the increase of the frequency of cecal and colonic contractions induced by the meal. Infused into the lateral hypothalamus at similar dosages, L364,718 had no effect on the postprandial enhancement of cecocolonic motility. Increase of cecocolonic spike-burst frequency induced by feeding or by cholecystokinin octapeptide injected into the ventromedial hypothalamus was abolished by previous intracerebroventricular but not intraperitoneal administration of atropine (1 microgram) and 4-diphenylacetoxy-N-methylpiperidine (1 microgram), a selective muscarinic M2-receptor antagonist. In contrast, pirenzepine (1 microgram, intracerebroventricularly) did not significantly reduce the meal- or cholecystokin octapeptide-induced increase in cecal and colonic motility. These results suggest that, in rats, (a) cholecystokinin octapeptide is involved in the generation of the cecocolonic motor response to a meal and these effects are mediated through cholecystokinin octapeptide receptors located in the ventromedial hypothalamic nuclei, and (b) these postprandial colonic motor changes involve central cholinergic activation through muscarinic M2 receptors.

Stress elicited by exposure to cold induces an increase of gastric emptying (GE) and intestinal transit of a caloric meal in mice and the release of corticotropin-releasing factor (CRF) and thyrotropin-releasing hormone (TRH) in the central nervous system (CNS). The present study proposed 1) to compare in mice the central effects of TRH, CRF, and cold-exposure stress on GE and intestinal transit of a caloric test meal consisting of 0.5 ml of reconstituted milk marked with 51Cr-labeled sodium chromate, and 2) using CRF and TRH antisera to determine whether TRH and CRF act in cascade or independently. The intracerebroventricular (icv) injection of TRH (0.5 microgram/kg) and CRF (1 microgram/kg), as well as cold stress, significantly increased GE, whereas 10-fold higher doses injected intraperitoneally were ineffective. The effect of cold stress on GE was abolished by prior icv injection of both CRF and TRH antisera. The effect of TRH was not blocked by CRF antiserum, but TRH antiserum suppressed the increase in GE induced by CRF. Moreover, both CRF and TRH antisera abolished changes in the rate of GE induced by exogenous CRF and TRH injection, respectively, therefore demonstrating the specific efficiency of immunoneutralization. CRF and cold stress both induced an increase in the rate of intestinal transit, while TRH had no effect. Antibodies to CRF prevented the intestinal stimulatory effect induced by CRF and cold stress. In contrast, antibodies to TRH were unable to antagonize either CRF or cold-stress induced increase in the rate of intestinal transit.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration of BN 52021 (50 mg/kg i.v.), a specific antagonist of platelet-activating factor (PAF), significantly reduced the intestinal myoelectric disturbances induced by E. coli endotoxin injection (50 micrograms/kg i.v.) by 62%. Thus, PAF may be involved in the intestinal motor alterations observed in endotoxic shock. When given in combination with indomethacin (10 mg/kg i.p.), BN 52021 inhibited endotoxic shock intestinal disturbances. Indomethacin alone also reduced PAF induced (25 micrograms/kg i.p.) disruption of migrating myoelectric complexes. Endotoxins may act on intestinal motility via release of endogenous PAF and prostaglandins, the effects of PAF being mediated through the release of prostaglandins.