The multicomponent medication Spascupreel attenuates stress-induced gut dysfunction in rats.

BACKGROUND: Irritable bowel syndrome (IBS) is a common disorder worldwide. It is characterized by abdominal pain/discomfort and changes in bowel habits. Due to the multifactorial pathophysiology and the heterogeneity of IBS patients, appropriate treatment of IBS is still a challenge. Spascupreel (SP-11), as a multicomponent medication, has the potential to modulate multiple pathophysiological pathways simultaneously. Therefore, the objective of the current study was to investigate the effects of oral SP-11 treatment on stress-induced changes of peripheral and central functions in a rat model mimicking human IBS. METHODS: Naïve Wistar rats were treated with SP-11 (0.9 tab/kg) or NaCl 0.9% by oral gavage for 4 days before 2-hour partial restraint stress (PRS) procedure. Twenty minutes after PRS, central and peripheral stress-induced changes affecting IBS were assessed. These include the hypothalamic-pituitary-adrenal (HPA) axis response through plasma ACTH and corticosterone measurements, visceral pain in response to colorectal distension, gut permeability, colonic mast cell number, and sensitization as well as gut transit time. RESULTS: Treatment with SP-11 reduced the HPA axis activation in response to PRS. At the gut level, a reduction in colonic hypersensitivity to colorectal distension, a normalization of gut transit time acceleration, a reduced mast cell sensitization, and a trend toward reduced gut hyperpermeability were observed. CONCLUSIONS: These data suggest that stress-induced IBS signs can be reduced using SP-11 in rats. The observed effects and the good tolerability of the drug make SP-11 an innovative candidate in the management of IBS.

Pharmacological, Pharmacokinetic, and Pharmacogenomic Aspects of Functional Gastrointestinal Disorders.

This article reviews medications commonly used for the treatment of patients with functional gastrointestinal disorders. Specifically, we review the animal models that have been validated for the study of drug effects on sensation and motility; the preclinical pharmacology, pharmacokinetics, and toxicology usually required for introduction of new drugs; the biomarkers that are validated for studies of sensation and motility endpoints with experimental medications in humans; the pharmacogenomics applied to these medications and their relevance to the FGIDs; and the pharmacology of agents that are applied or have potential for the treatment of FGIDs, including psychopharmacologic drugs.

A pilot study on faecal MMP-9: a new noninvasive diagnostic marker of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading malignancies worldwide, therefore cheap noninvasive screening methods are of great importance. Matrix-metalloproteinase-9 (MMP-9) has a role in the progression of CRC, and its level is elevated in tumour biopsies. Faecal MMP-9 levels are increased in active ulcerative colitis patients, but in CRC patients, they have never been measured. We aimed to assess the faecal MMP-9 levels in patients undergoing total colonoscopy according to endoscopic and histological diagnosis. METHODS: One hundred and nine patients provided faecal samples for MMP-9 analysis. A total colonoscopy was performed; suspicious lesions were evaluated by histology. Faecal MMP-9 levels were measured by ELISA. RESULTS: The number of patients allocated to different groups were: negative/diverticulosis: 34 (referred to as controls); hyperplastic polyps: 15; adenomas: 32 (22 at high risk); and CRC: 28. Faecal MMP-9 was significantly increased in CRC compared with all other groups (P<0.001). Faecal MMP-9 was suitable to distinguish CRC patients from controls (sensitivity: 89.3%; specificity: 91.2%). By means of a lower cutoff level, faecal MMP-9 identified high-risk adenomas besides CRC (sensitivity: 76%; specificity: 85.3%). This lower cutoff level screened 59% of high-risk adenomas. CONCLUSIONS: Faecal MMP-9 may be a promising new noninvasive marker in CRC.

Inhibitory effects of SKI3246, the rhizome extract of Atractylodes japonica, on visceral hypersensitivity in experimental irritable bowel syndrome rat models.

We evaluated the effect of SKI3246, the 50% ethanol extract of the rhizome of Atractylodes japonica, on visceral hypersensitivity, which is a major characteristic feature of IBS. We used various rat models of visceral hypersensitivity to assess the visceral pain responses to colorectal distension (CRD) in comparison with conventional IBS treatments. Oral administration of SKI3246 dose-dependently and significantly attenuated the abdominal withdrawal reflex (AWR) score in a model of acetic acid-induced visceral hypersensitivity. We also found that it reduced the number of abdominal contractions in response to CRD in a model of 2,4,6-trinitrobenzenesulfonic acid-induced visceral hypersensitivity, which was comparable to ramosetron or alosetron. Furthermore, treatment with SKI3246 also increased the pain threshold and abolished the elevated AWR scores to CRD in a rat model of neonatal maternal separation. We presumed that the modulation of the NK2 receptor is involved in the inhibitory activity of SKI3246 on the basis that it significantly inhibited the contraction of the distal colonic muscle induced by neurokinin A, the NK2 receptor agonist. The present results indicate that SKI3246 has the potential to be an effective therapeutic agent for IBS, especially insofar as it can relieve visceral hypersensitivity.

A concomitant treatment by CO2-releasing suppositories improves the results of anorectal biofeedback training in patients with dyschezia: results of a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Dyschezia is a defecatory disorder that places a heavy burden on a patient's quality of life. Biofeedback is the recommended treatment in most cases. OBJECTIVE: The objective of our study was to test whether a CO2-releasing suppository for patients with dyschezia could be effective in improving biofeedback training results. DESIGN: A randomized, double-blind, multicenter, placebo-controlled study was conducted in patients (18-75 years of age) with dyschezia defined according to the modified Rome III criteria. Patients were randomly assigned to either a CO2-releasing suppository or placebo suppository once per day for 21 days. SETTINGS: This was a multicenter trial. PATIENTS: A total of 122 patients were randomly assigned (62 intervention group and 60 placebo group). MAIN OUTCOME MEASURES: The primary end point was the change from day 0 to day 21 in intensity of symptoms on the basis of a self-assessed dyschezia using a visual analog scale (range, 0-100). Analyses were performed using intention-to-treat principles. RESULTS: A greater reduction from baseline to day 21 in symptom visual analog scale score was observed in the intervention group (-41.3 mm) than in the control group (-22.3 mm). Some secondary efficacy parameters improved more in the intervention group, including the percentage of patients who improved ≥50%, symptom intensity over 21 days, stool stains on underwear or pads, and need to practice manual maneuvers to facilitate defecation at day 21. At day 21, rectal sensitivity in the intervention group (31.4 mL) was lower than in the control group (39.1 mL). LIMITATIONS: There was a lower number of patients recruited than planned by the protocol. The sponsor stopped the trial before the inclusion of 306 participants, with no intermediate analysis. In addition, the main analysis conducted on the full analysis set population could have led to a statistical bias. CONCLUSIONS: The results of this multicenter trial demonstrate the added benefits of a CO2-releasing suppository in patients with dyschezia who were treated by anorectal biofeedback training.

Fecal protease activity is associated with compositional alterations in the intestinal microbiota.

OBJECTIVE: Intestinal proteases carry out a variety of functions in the gastrointestinal (GI) tract. Studies have reported that elevated enteric proteases in patients with GI disease can alter intestinal physiology, however the origin (human vs. microbial) of elevated proteases in patients with GI disease is unclear. AIM: The aim of this study was to investigate the association between protease activity and the microbiota in human fecal samples. DESIGN: In order to capture a wide range of fecal protease (FP) activity stool samples were collected from 30 IBS patients and 24 healthy controls. The intestinal microbiota was characterized using 454 high throughput pyro-sequencing of the 16S rRNA gene. The composition and diversity of microbial communities were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. FP activity levels were determined using an ELISA-based method. FP activity was ranked and top and bottom quartiles (n=13 per quartile) were identified as having high and low FP activity, respectively. RESULTS: The overall diversity of the intestinal microbiota displayed significant clustering separation (p = 0.001) between samples with high vs. low FP activity. The Lactobacillales, Lachnospiraceae, and Streptococcaceae groups were positively associated with FP activity across the entire study population, whilst the Ruminococcaceae family and an unclassified Coriobacteriales family were negatively associated with FP activity. CONCLUSIONS: These data demonstrate significant associations between specific intestinal bacterial groups and fecal protease activity and provide a basis for further causative studies investigating the role of enteric microbes and GI diseases.

Gastrointestinal pain: unraveling a novel endogenous pathway through uroguanylin/guanylate cyclase-C/cGMP activation.

The natural hormone uroguanylin regulates intestinal fluid homeostasis and bowel function through activation of guanylate cyclase-C (GC-C), resulting in increased intracellular cyclic guanosine-3',5'-monophosphate (cGMP). We report the effects of uroguanylin-mediated activation of the GC-C/cGMP pathway in vitro on extracellular cGMP transport and in vivo in rat models of inflammation- and stress-induced visceral hypersensitivity. In vitro exposure of intestinal Caco-2 cells to uroguanylin stimulated bidirectional, active extracellular transport of cGMP into luminal and basolateral spaces. cGMP transport was significantly and concentration dependently decreased by probenecid, an inhibitor of cGMP efflux pumps. In ex vivo Ussing chamber assays, uroguanylin stimulated cGMP secretion from the basolateral side of rat colonic epithelium into the submucosal space. In a rat model of trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity, orally administered uroguanylin increased colonic thresholds required to elicit abdominal contractions in response to colorectal distension (CRD). Oral administration of cGMP mimicked the antihyperalgesic effects of uroguanylin, significantly decreasing TNBS- and restraint stress-induced visceromotor response to graded CRD in rats. The antihyperalgesic effects of cGMP were not associated with increased colonic spasmolytic activity, but were linked to significantly decreased firing rates of TNBS-sensitized colonic afferents in rats in response to mechanical stimuli. In conclusion, these data suggest that the continuous activation of the GC-C/cGMP pathway along the intestinal tract by the endogenous hormones guanylin and uroguanylin results in significant reduction of gastrointestinal pain. Extracellular cGMP produced on activation of GC-C is the primary mediator in this process via modulation of sensory afferent activity.

Luminal cysteine-proteases degrade colonic tight junction structure and are responsible for abdominal pain in constipation-predominant IBS.

OBJECTIVES: Luminal serine-proteases lead to increased colonic paracellular permeability and visceral hypersensitivity in patients with diarrhea-predominant irritable bowel syndrome (IBS-D). Other proteases, namely cysteine-proteases (CPs), increase airway permeability by digesting epithelial tight junction proteins. In this study, we focused on constipation-predominant IBS (IBS-C) and we aimed to (i) evaluate CP levels in two cohorts of IBS patients, (ii) test if IBS-C fecal supernatant (FSN) affects permeability, and visceral sensitivity after repeated administrations in mice, and (iii) evaluate occludin expression in IBS-C colonic biopsies. METHODS: Fecal CP activity was determined using selective substrate and inhibitor (E64). The effect of papain, as positive control, and IBS-C FSN administrations were evaluated on colonic paracellular permeability and mucosal occludin levels in mice and T84 monolayers. Occludin protein levels were evaluated in IBS-C colonic biopsies. Sensitivity to colorectal distension (CRD) was measured after repeated administrations of IBS-C FSN. RESULTS: We found in a subset of IBS-C patients an enhanced fecal CP activity, in comparison with healthy controls and IBS-D patients. CP activity levels positively correlated with disease severity and abdominal pain scoring. This association was confirmed by receiver operating characteristic curve analysis. In mice, repeated application of IBS-C FSN into colon triggered increased permeability, linked to the enzymatic degradation of occludin, and was associated with enhanced visceral sensitivity to CRD. Finally, occludin levels were found decreased in colonic biopsies from IBS-C patients, and IBS-C FSNs were able to degrade recombinant human occludin in vitro. All these effects were abolished by preincubation of IBS-C FSN with a CP inhibitor, E64. CONCLUSIONS: These data suggest that luminal CPs may represent a new factor contributing to the genesis of symptoms in IBS.

Ocular inflammation and corneal permeability alteration by benzalkonium chloride in rats: a protective effect of a myosin light chain kinase inhibitor.

PURPOSE: The aim of this study was to evaluate the interest of an ophthalmic eyedrop preparation containing a myosin light chain kinase (MLCK) inhibitor, ML-7, in the treatment of ocular surface. The local protective effect on the inflammation and the increase of corneal permeability induced by benzalkonium (BAK) was evaluated. METHODS: An ocular instillation of 10 lL BAK at a concentration of 0.1% in PBS was performed on rats. The eyes were rinsed with sterilized water, 10 minutes after BAK preceded by instillation at T -24, -12, and -0.5 hours of 10 lL ofML-7: 100 µg (10 µL) into a gel form vehicle. All animals were sacrificed 6 hours after BAK instillation. The eyes were isolated for study in a masked manner. The ocular surface inflammation was assessed by measuring the inflammatory cell infiltration by a histologic quantitative analysis and for total ocular myeloperoxidase (MPO) activity. The tight junction permeability was tested. RESULTS: Instillation of 0.1% BAK increased the inflammation of the eye. The quantitative analysis showed an increase in the number of eosinophil and neutrophil polynuclears, and MPO activity. Pretreatment with ML-7 reduced inflammation (P < 0.05). The vehicle alone produced no notable effects. BAK instillation also thickened the fluorescent corneal front on frozen sections, indicating an increase of tight junction permeability. Pretreatment with ML-7 suppressed BAK-induced alterations of paracellular permeability while the vehicle had no visible effects. CONCLUSIONS: Our study indicates that the inhibition of corneal cytoskeleton contraction by an MLCK inhibitor prevents BAK-induced ocular inflammatory response, and that ML-7 may be a new and original preparation in the treatment of ocular surface pathologies.

Influence of simethicone and alverine on stress-induced alterations of colonic permeability and sensitivity in rats: beneficial effect of their association.

OBJECTIVES: Alverine, an antispasmodic agent for the treatment of irritable bowel syndrome (IBS), may be combined with simethicone, a protective agent of the mucosa. Stress is a major factor triggering abdominal pain in IBS and causing hypersensitivity to colonic distension in animals through an increased colonic permeability. The antinociceptive effects of alverine and simethicone, separately or in association, were evaluated on stress-induced colonic hypersensitivity to distension in rats. The influence of simethicone on altered permeability was also tested. METHODS: Groups of 8-10 female adult Wistar rats (200-250 g) housed individually were used. Gut paracellular permeability was evaluated after 2 h of partial restraint stress using oral gavage with 5¹Cr-EDTA and 24 h of urine collection. The number of abdominal cramps during colonic distension was evaluated in animals equipped with electrodes on their abdominal striated muscles. KEY FINDINGS: At 200 mg/kg p.o. twice a day, but not at lower doses, simethicone reduced stress-induced increase of colonic permeability and hypersensitivity to distension. Administered alone at 10 mg/kg p.o., alverine also reduced stress-induced hypersensitivity to distension; lower doses were inactive. However, alverine administered at an inactive dose with simethicone suppressed stress-induced hypersensitivity to distension. CONCLUSIONS: We conclude that both simethicone and alverine have visceral antinociceptive effects by two different mechanisms and that simethicone exerts a potentiating effect on the antinociceptive action of alverine.

Protective and worsening peripheral nociceptin/orphanin FQ receptor-mediated effect in a rat model of experimental colitis.

Nociceptin/orphanin FQ (N/OFQ) and nociceptin orphanin peptide (NOP) receptors represent an endogenous system modulating gastrointestinal functions and inflammation. We investigated the peripheral effect of N/OFQ and of UFP-101, the NOP antagonist, in a model of colitis induced by TNBS (2,4,6 trinitrobenzenesulphonic acid; 60mg/kg). Male rats received two intraperitoneal injections per day of N/OFQ, UFP-101 or saline for 3 days after colitis induction. Four days after TNBS, animals were sacrificed and colonic histological damage, myeloperoxidase (MPO) activity and cytokine (IL-1ß and IL-10) levels were evaluated. N/OFQ plasmatic levels were assessed by radioimmunoassay. TNBS increased all the inflammatory variables considered. In colitic rats, N/OFQ (0.02 and 0.2nmol/kg) improved microscopic damage, MPO activity and decreased IL-1ß levels in comparison with TNBS group, whereas at the highest dose (20nmol/kg) the peptide worsened colitis. UFP-101 at the dose of 1nmol/kg, without pharmacological activity, antagonised the protective effect of N/OFQ (0.2nmol/kg) on colitis, but at a dose level of 3 and 10nmol/kg worsened inflammation, revealing the endogenous N/OFQergic system protective role. N/OFQ plasmatic levels were not modified in TNBS-treated rats compared with controls, whereas they were reduced in rats treated with the doses of UFP-101 aggravating colitis. In conclusion, peripheral low doses of N/OFQ have a beneficial effect on colonic inflammation in rats. In contrast, N/OFQ at a dose 100-1000-fold higher than those that protect worsens colitis, probably through different mechanisms. The peripheral N/OFQergic system can represent a new field of investigation in some intestinal inflammatory conditions.

Fecal MMP-9: a new noninvasive differential diagnostic and activity marker in ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is characterized by frequent relapses, with the presence of colorectal inflammation and mucosal lesions. Matrix-metalloprotease 9 (MMP-9) is elevated in colonic biopsies, urine, and blood plasma of UC patients. MMP-9 has been suggested as a predictor of UC in the urine of children; however, 20% of the controls tested positive. So far, fecal MMP-9 levels have never been measured. Our aims were: 1) to compare fecal MMP-9 levels in UC patients to control subjects and a functional gastrointestinal disorder characterized by diarrhea (IBS-D); 2) to test the correlation between UC disease activity and fecal levels of MMP-9; and 3) to correlate fecal MMP-9 levels with a known fecal marker of UC activity, calprotectin. METHODS: UC (n = 47), IBS-D (n = 23) patients, and control subjects (n = 24) provided fecal samples for MMP-9 analysis. In UC patients, disease severity was evaluated by the Mayo score. Fecal MMP-9 and calprotectin levels were measured by enzyme-linked immunosorbent assay and lateral flow assay, respectively. RESULTS: MMP-9 was undetectable or ≤0.22 ng/mL in the feces of all controls and IBS-D patients. In UC patients, fecal MMP-9 levels significantly correlated with the overall Mayo score (P < 0.001), the endoscopic score (P < 0.001), and the serum C-reactive protein levels (P = 0.002). Additionally, in UC patients fecal MMP-9 levels showed a significant correlation with a known disease activity marker, fecal calprotectin (P = 0.014). CONCLUSIONS: These results highlight fecal MMP-9 as a useful tool in the differential diagnosis of diarrheic disorders and in the noninvasive evaluation of disease activity and mucosal healing in UC.

Prevention of gut leakiness by a probiotic treatment leads to attenuated HPA response to an acute psychological stress in rats.

BACKGROUND AND AIMS: Intestinal barrier impairment is incriminated in the pathophysiology of intestinal gut disorders associated with psychiatric comorbidity. Increased intestinal permeability associated with upload of lipopolysaccharides (LPS) translocation induces depressive symptoms. Gut microbiota and probiotics alter behavior and brain neurochemistry. Since Lactobacillus farciminis suppresses stress-induced hyperpermeability, we examined whether (i) L. farciminis affects the HPA axis stress response, (ii) stress induces changes in LPS translocation and central cytokine expression which may be reversed by L. farciminis, (iii) the prevention of "leaky" gut and LPS upload are involved in these effects. METHODS: At the end of the following treatments female rats were submitted to a partial restraint stress (PRS) or sham-PRS: (i) oral administration of L. farciminis during 2 weeks, (ii) intraperitoneal administration of ML-7 (a specific myosin light chain kinase inhibitor), (iii) antibiotic administration in drinking water during 12 days. After PRS or sham-PRS session, we evaluated LPS levels in portal blood, plasma corticosterone and adrenocorticotropic hormone (ACTH) levels, hypothalamic corticotropin releasing factor (CRF) and pro-inflammatory cytokine mRNA expression, and colonic paracellular permeability (CPP). RESULTS: PRS increased plasma ACTH and corticosterone; hypothalamic CRF and pro-inflammatory cytokine expression; CPP and portal blood concentration of LPS. L. farciminis and ML-7 suppressed stress-induced hyperpermeability, endotoxemia and prevented HPA axis stress response and neuroinflammation. Antibiotic reduction of luminal LPS concentration prevented HPA axis stress response and increased hypothalamic expression of pro-inflammatory cytokines. CONCLUSION: The attenuation of the HPA axis response to stress by L. farciminis depends upon the prevention of intestinal barrier impairment and decrease of circulating LPS levels.

Leaky gut in patients with diarrhea-predominant irritable bowel syndrome and inactive ulcerative colitis.

BACKGROUND/AIMS: Defective epithelial barrier has been implicated in the pathogenesis of irritable bowel syndrome (IBS) and inflammatory bowel diseases. The aim of this study was to investigate gut permeability in patients with inactive ulcerative colitis (UC) and in patients with IBS. METHODS: IBS patients of the diarrhea-predominant (IBS-D) and of the constipation-predominant subgroup (IBS-C), patients with inactive UC and healthy subjects were enrolled. Gut permeability was evaluated by measuring 24-hour urine excretion of orally administered (51)Cr-EDTA. Clinical symptoms were evaluated in IBS-D patients and correlated to colonic permeability. RESULTS: There was a significant decrease in the proximal small intestinal permeability in IBS-C patients compared to controls (0.26 ± 0.05 vs. 0.63 ± 0.1%; p < 0.05). Distal small intestinal permeability showed no significant difference in the studied group of patients compared to controls. Colonic permeability of IBS-D and inactive UC patients was significantly increased compared to controls (2.68 ± 0.35 and 3.74 ± 0.49 vs. 1.04 ± 0.18%; p < 0.05, p < 0.001). Colonic permeability of IBS-D patients correlated with stool frequency. CONCLUSIONS: Elevated gut permeability is localized to the colon both in IBS-D and in inactive UC patients.

Intracolonic infusion of fecal supernatants from ulcerative colitis patients triggers altered permeability and inflammation in mice: role of cathepsin G and protease-activated receptor-4.

BACKGROUND: Cathepsin G (Cat-G) is a neutrophil serine-protease found in the colonic lumen of ulcerative colitis (UC) patients. Cat-G is able to activate protease-activated receptor-4 (PAR(4) ) located at the apical side of enterocytes, leading to epithelial barrier disruption. However, the mechanisms through which Cat-G triggers inflammation are not fully elucidated. The aims of our study were to evaluate in vivo the effects of UC fecal supernatants and Cat-G on epithelial barrier function and inflammation, and the connection between these two parameters. METHODS: Male balb/c mice were used in this study. We evaluated the effect of a 2-hour intracolonic infusion of 1) fecal supernatants from UC patients pretreated or not with specific Cat-G inhibitor (SCGI); 2) PAR(4) -activating peptide (PAR(4) -AP); and 3) Cat-G on colonic myeloperoxidase (MPO) activity and paracellular permeability (CPP). The involvement of PAR(4) was assessed by pretreating animals with pepducin P4pal-10, which blocks PAR(4) signaling. We investigated the role of myosin light chain (MLC) kinase by using its inhibitor, ML-7, and we determined phosphorylated MLC (pMLC) levels in mice colonic mucosa. RESULTS: UC fecal supernatants, Cat-G, and PAR(4) agonist increased both CPP and MPO activity in comparison with healthy subjects fecal supernatants. ML-7 inhibited the CPP increase triggered by Cat-G by 92.3%, and the enhanced MPO activity by 43.8%. Intracolonic infusion of UC fecal supernatant determined an increased phosphorylation level of MLC. CONCLUSIONS: These observations support that luminal factors such as Cat-G play an important proinflammatory role in the pathogenesis of colitis, mainly depending on CPP increase by MLC phosphorylation.

Proinflammatory role of vasopressin through V1b receptors in hapten-induced experimental colitis in rodents: implication in IBD.

Vasopressin and its receptors modulate several gut functions, but their role in intestinal inflammation is unknown. Our aims were to determine 1) the localization of V1b receptors in human and rodent colon, 2) the role of vasopressin and V1b receptors in experimental colitis using two approaches: V1b⁻(/)⁻ mice and a selective V1b receptor antagonist, SSR149415, and 3) the mechanisms involved. V1b receptors were localized in normal and inflamed colon from humans and rats. Experimental colitis was induced in rats and mice and some groups were treated before or after colitis induction with oral SSR149415 (3-30 mg/kg). Other groups of mice were submitted to dehydration to increase vasopressin plasma levels, prior to colitis induction. Body weight, damage scores, MPO, and TNF-α tissue levels were determined. Finally, colonic segments of wild-type (WT) and V1b⁻(/)⁻ mice were mounted in Ussing chambers and paracellular permeability in response to vasopressin was studied. V1b receptors were expressed in enterocytes and ganglia cells of the enteric nervous system of human and rat intestine. Expression levels were independent from inflammatory status. Colitis was less severe in rodents treated by either preventive or curative SSR149415 and in V1b⁻(/)⁻ mice. 2,4,6-Trinitrobenzene sulfonic acid induced a strong mortality in dehydrated animals that was reversed by preventive SSR149415 or mast cell stabilizer. Vasopressin significantly increased paracellular permeability in WT, but not in V1b⁻(/)⁻ mice. Preincubation of colon tissues with SSR149415 abolished the vasopressin effect. Similarly, vasopressin had no effect in colonic preparations from WT mice pretreated with mast cell stabilizers. Vasopressin, through V1b receptor interaction, has proinflammatory properties linked to mast cell activation and downstream alterations of the colonic epithelial barrier. These findings underline the potential interest of V1b receptor blockers in gut inflammatory diseases.

Evidence of central and peripheral sensitization in a rat model of narcotic bowel-like syndrome.

BACKGROUND & AIMS: Narcotic bowel syndrome (NBS) is a subset of opioid bowel dysfunctions that results from prolonged treatment with narcotics and is characterized by chronic abdominal pain. NBS is under-recognized and its molecular mechanisms are unknown. We aimed to (1) develop a rat model of NBS and (2) to investigate its peripheral and central neurobiological mechanisms. METHODS: Male Wistar rats were given a slow-release emulsion that did or did not contain morphine (10 mg/kg) for 8 days. Visceral sensitivity to colorectal distension (CRD) was evaluated during and after multiple administrations of morphine or vehicle (controls). The effects of minocycline (a microglia inhibitor), nor-binaltorphimine (a kappa-opioid antagonist), and doxantrazole (a mast-cell inhibitor) were observed on morphine-induced visceral hyperalgesia. Levels of OX-42, P-p38 mitogen-activated protein kinase, rat mast cell protease II, and protein gene product 9.5 were assessed at different spinal segments (lumbar 6 to sacral 1) or colonic mucosa by immunohistochemistry. RESULTS: On day 8 of morphine administration, rats developed visceral hyperalgesia to CRD (incipient response) that lasted for 8 more days (delayed response). Minocycline reduced the incipient morphine-induced hypersensitivity response to CRD whereas nor-binaltorphimine and doxantrazole antagonized the delayed hyperalgesia. Levels of OX-42 and P-p38 increased in the spinal sections, whereas rat mast cell protease II and protein gene product 9.5 increased in the colonic mucosa of rats that were given morphine compared with controls. CONCLUSIONS: We developed a rat model of narcotic bowel-like syndrome and showed that spinal microglia activation mediates the development of morphine-induced visceral hyperalgesia; peripheral neuroimmune activation and spinal dynorphin release represent an important mechanism in the delayed and long-lasting morphine-induced colonic hypersensitivity response to CRD.

Luminal cathepsin g and protease-activated receptor 4: a duet involved in alterations of the colonic epithelial barrier in ulcerative colitis.

Impairment of the colonic epithelial barrier and neutrophil infiltration are common features of inflammatory bowel disease. Luminal proteases affect colonic permeability through protease-activated receptors (PARs). We evaluated: (i) whether fecal supernatants from patients with ulcerative colitis (UC) trigger alterations of colonic paracellular permeability and inflammation, and (ii) the roles of cathepsin G (Cat-G), a neutrophil serine protease, and its selective receptor, PAR(4), in these processes. Expression levels of both PAR(4) and Cat-G were determined in colonic biopsies from UC and healthy subjects. The effects of UC fecal supernatants on colonic paracellular permeability were measured in murine colonic strips. Involvement of Cat-G and PAR(4) was evaluated using pepducin P4pal-10 and specific Cat-G inhibitor (SCGI), respectively. In addition, the effect of PAR(4)-activating peptide was assessed. UC fecal supernatants, either untreated or pretreated with SCGI, were infused into mice, and myeloperoxidase activity was determined. PAR(4) was found to be overexpressed in UC colonic biopsies. Increased colonic paracellular permeability that was triggered by UC fecal supernatants was blocked by both SCGI (77%) and P4pal-10 (85%). Intracolonic infusion of UC fecal supernatants into mice increased myeloperoxidase activity. This effect was abolished by SCGI. These observations support that both Cat-G and PAR(4) play key roles in generating and/or amplifying relapses in UC and provide a rationale for the development of new therapeutic agents in the treatment of this disease.

Fecal proteases from diarrheic-IBS and ulcerative colitis patients exert opposite effect on visceral sensitivity in mice.

Elevated colonic luminal serine-protease (Ser-P) activity of diarrhea-predominant IBS (IBS-D) patients evokes a proteinase-activated receptor (PAR)-2-mediated colonic hypersensitivity in mice. Despite similarly elevated Ser-P levels in feces, patients with IBD exhibit visceral hypo- or normosensitivity to rectal distension, as opposed to IBS-D. To explain these discrepancies we studied the effect of colonic infusion of fecal supernatants from ulcerative colitis (UC) patients to colorectal mechanical sensitivity of mice and explored the involvement of PAR-4 and its activator Cathepsin-G (Cat-G). Fecal protease activities were assayed in healthy subjects, IBS-D and UC patients in presence or not of antiproteases or Cat-G inhibitor. Following intracolonic infusion of fecal supernatants from healthy subjects, IBS-D and UC patients or PAR-4 activating peptide (PAR-4-AP) or Cat-G, EMG response to colorectal balloon distension was recorded in mice. This nociceptive response was also determined after treatment with pepducin (PAR-4 antagonist) on UC supernatant or after a preincubation with antiproteases or Cat-G inhibitor. In contrast to IBS-D supernatant, UC supernatant promoted colonic hyposensitivity to distension, an effect mimicked by PAR-4-AP or Cat-G. UC supernatant-induced hypoalgesia was inhibited by a cocktail of antiproteases. However, blockade of PAR-4 or Cat-G inhibition resulted in colonic hypersensitivity similar to that observed after IBS-D supernatant infusion. Despite similarly elevated Ser-P activities, IBS-D and UC fecal supernatant display visceral pro- and antinociceptive effects in mice, respectively. Visceral hyposensitivity induced by fecal supernatant from UC patients results from PAR-4 activation by cathepsin-G, counterbalancing the pronociceptive effect of simultaneous PAR-2 activation.

Oestradiol decreases colonic permeability through oestrogen receptor beta-mediated up-regulation of occludin and junctional adhesion molecule-A in epithelial cells.

Oestradiol modulates paracellular permeability and tight junction (TJ) function in endothelia and reproductive tissues, but whether the ovarian hormones and cycle affect the paracellular pathway in the intestinal epithelium remains unclear. Oestrogen receptors (ERs) are expressed in intestinal epithelial cells, and oestradiol regulates epithelium formation. We examined the effects of oestrous cycle stage, oestradiol benzoate (EB), and progesterone (P) on colonic paracellular permeability (CPP) in the female rat, and whether EB affects expression of the TJ proteins in the rat colon and the human colon cell line Caco-2. In cyclic rats, CPP was determined through lumen-to-blood (51)Cr-labelled EDTA clearance, and in Ussing chambers for dextran permeability. CPP was also examined in ovariectomized (OVX) rats treated with P or EB, with and without the ER antagonist ICI 182,780, or with the selective agonists for ER beta (propyl pyrazole triol; PPT) or ER beta (diarylpropionitrile; DPN). In oestrus rats, CPP was reduced (P < 0.01) relative to dioestrus. In OVX rats, EB dose-dependently decreased CPP, an effect mimicked by DPN and blocked by ICI 182,780, whereas P had no effect. Oestradiol increased occludin mRNA and protein in the colon (P < 0.05), but not zona occludens (ZO)-1. Further, EB and DPN enhanced occludin and junctional adhesion molecule (JAM)-A expression in Caco-2 cells without change in ZO-1, an effect blocked by ICI 182,780. These data show that oestrogen reinforces intestinal epithelial barrier through ER beta-mediated up-regulation of the transmembrane proteins occludin and JAM-A determining paracellular spaces. These findings highlight the importance of the ER beta pathway in the control of colonic paracellular transport and mucosal homeostasis.