R. Garcia-Villar

Acute and chronic stress differently affect visceral sensitivity to rectal distension in female rats

Stressful life events are frequently associated with outward signs of irritable bowel syndrome (IBS). Increasing evidence suggests that acute and chronic stress stimuli implicate different physiological mechanisms and neuroendocrine responses. Therefore, we investigated the influence of acute and chronic stress on visceral nociception in female rats and the involvement of colonic mast cells in this effect. The effect of acute and chronic partial restraint stress (PRS) on visceral sensitivity to rectal distension (RD) was assessed by abdominal muscle electromyography. Colonic mast cell activation was determined by measuring histamine release after in vitro stimulation with substance P (SP) in colonic samples from rats experiencing RD vs. controls. Acute PRS significantly enhanced abdominal response to RD compared with sham PRS for all volumes of distension. In contrast, chronic PRS induced a hyperalgesic response for the highest volumes of distension (0.8 and 1.2 mL), but did not affect the number of abdominal contractions for the lowest volume (0.4 mL) compared with controls. Both acute and chronic PRS increased in vitro SP‐induced histamine release without affecting mast cell numbers. RD induced similar in vitro histamine release from colonic samples from both acute and chronic PRS rats; this release, however, was significantly higher than that measured in sham‐PRS rats. Acute and chronic PRS differently influence visceral sensitivity in response to RD in female rats. This difference, however, cannot be attributed to a different effect of either stress paradigm on mast cell histamine release.

Colonic luminal proteases activate colonocyte proteinase-activated receptor-2 and regulate paracellular permeability in mice.

Abstract  Luminal activation of protease‐activated receptors‐2 (PAR2) on colonocytes by trypsin or PAR2‐activating peptide increases colonic paracellular permeability (CPP). The aim of this study was to evaluate the role of proteases from endogenous and bacterial origin in the modulation of CPP and colonocyte PAR2 expression in mice. CPP was assessed with 51Cr‐EDTA after intracolonic administration of different protease inhibitors. After 12 days of oral antibiotic treatment, measurements of colonic luminal serine protease activity (CLSPA), CPP, mucosal mouse mast cell proteinase‐1 (MMCP‐1) content, immunochemistry of PAR2 and assessment of effects of PAR2 agonist (SLIGRL) and mast cell degranulator (C48/80) on CPP in Ussing chambers were performed. Immunochemistry was repeated after intracolonic trypsin administration. Colonic infusion of protease inhibitors significantly reduced CPP. In antibiotic‐treated mice, CLSPA was reduced coupled with a decrease in PAR2 expression, but with no change in CPP and MMCP‐1 content. Trypsin administration restored PAR2 expression. The increase in CPP induced by SLIGRL and C48/80 was reduced after antibiotic treatment. Protease activity of colonic content plays an important role in the regulation of mucosal barrier through activation of PAR2.

Effects of tachykinin receptor antagonists on the rat jejunal distension pain response

Distension of the rat intestine causes a cardiovascular response which is indicative of nociception. Since tachykinins are involved in nociception, we tested the effect of neurokinin receptor antagonists against the distension-induced response. The jejunal distension-induced depressor responses were inhibited in a dose-dependent fashion by CP 99,994 (+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine, tachykinin NK1 receptor antagonist, ED50 = 0.8 mg/kg) and SR 48968 (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichloropheny l)butyl]benzamide, tachykinin NK2 receptor antagonist, ED50 = 0.7 mg/kg). SR 142801 (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)prop yl)-4-phenylpiperidin-4-yl)-N-methylacetamide, tachykinin NK3 receptor antagonist, 0.3-10 mg/kg) did not significantly affect the depressor responses to jejunal distension. In addition, CP 99,994 (3 mg/kg) and SR 48968 (3 and 10 mg/kg) reduced sensitivity to distension as revealed by a 2.7-fold (CP 99.994, 3 mg/kg), 2.6-fold (SR 48968, 3 mg/kg) and 4.7-fold (SR 48968, 10 mg/kg) increase in the threshold pressure. Intestinal compliance was not affected by the antagonists. In conclusion, these results suggest that tachykinin NK1 and NK2 but not NK3 receptors are possibly involved in the rat jejunal distension pain response.

Dexamethasone prevents visceral hyperalgesia but not colonic permeability increase induced by luminal protease - activated receptor-2 agonist in rats

Background: Low-grade inflammation may play a role in the pathogenesis of irritable bowel syndrome (IBS). Although corticosteroids are potent inhibitors of inflammatory processes, only one study with corticosteroids in patients with postinfectious IBS exists, which suggests that prednisolone is not an effective treatment for IBS symptoms. Aim: To evaluate whether dexamethasone treatment prevents protease-activated receptor-2 (PAR-2) activation-induced visceral hyperalgesia and increased permeability in rats, and to determine whether the effects involve colonic mast cells. Methods: Abdominal contractions provoked by rectal distension were recorded in rats equipped with intramuscular electrodes. Changes in visceral hypersensitivity provoked by intracolonic administration of PAR-2-activating peptide (SLIGRL; H-serine-leucine-isoleucine-glycine-arginine-leucine-OH), changes in colonic mucosal rat mast cell protease-II (RMCP-II) content, mast cell count and PAR-2 expression were measured after a 4-day treatment with dexamethasone (1 mg/day/rat intraperitoneally) or its vehicle (water). The effect of mast cell stabiliser (doxantrazole, 1 mg/kg intraperitoneally, 2 h before and 6 h after intracolonic infusion of SLIGRL) on SLIGRL-induced visceral hyperalgesia was also assessed. The effects of SLIGRL and a mast cell degranulator (compound 48/80) on the permeability of colonic strips from vehicle- or dexamethasone-treated rats were investigated in Ussing chambers. Results: 4 days of dexamethasone as well as doxantrazole diminished the SLIGRL-induced hyperalgesia for all volumes of distension. This effect of dexamethasone was accompanied by a reduced responsiveness of colonic permeability to compound 48/80, and decreased RMCP-II content and mast cell number. Dexamethasone treatment did not influence colonic mucosal PAR-2 expression and permeability responsiveness to SLIGRL. Conclusions: Dexamethasone treatment improves PAR-2 agonist-induced visceral hypersensitivity but does not prevent PAR-2 agonist-induced increase in colonic permeability in rats. This effect is coupled with a reduction of colonic mast cell number and RMCP-II contents.

Perinatal Exposure to a Low Dose of Bisphenol A Impaired Systemic Cellular Immune Response and Predisposes Young Rats to Intestinal Parasitic Infection

Perinatal exposure to the food contaminant bisphenol A (BPA) in rats induces long lasting adverse effects on intestinal immune homeostasis. This study was aimed at examining the immune response to dietary antigens and the clearance of parasites in young rats at the end of perinatal exposure to a low dose of BPA. Female rats were fed with BPA [5 µg/kg of body weight/day] or vehicle from gestational day 15 to pup weaning. Juvenile female offspring (day (D)25) were used to analyze immune cell populations, humoral and cellular responses after oral tolerance or immunization protocol to ovalbumin (OVA), and susceptibility to infection by the intestinal nematode Nippostrongylus brasiliensis (N. brasiliensis). Anti-OVA IgG titers following either oral tolerance or immunization were not affected after BPA perinatal exposure, while a sharp decrease in OVA-induced IFNγ secretion occurred in spleen and mesenteric lymph nodes (MLN) of OVA-immunized rats. These results are consistent with a decreased number of helper T cells, regulatory T cells and dendritic cells in spleen and MLN of BPA-exposed rats. The lack of cellular response to antigens questioned the ability of BPA-exposed rats to clear intestinal infections. A 1.5-fold increase in N. brasiliensis living larvae was observed in the intestine of BPA-exposed rats compared to controls due to an inappropriate Th1/Th2 cytokine production in infected jejunal tissues. These results show that perinatal BPA exposure impairs cellular response to food antigens, and increases susceptibility to intestinal parasitic infection in the juveniles. This emphasized the maturing immune system during perinatal period highly sensitive to low dose exposure to BPA, altering innate and adaptative immune response capacities in early life.

Alterations of intestinal motor responses to various stimuli after Nippostrongylus brasiliensis infection in rats: role of mast cells

Nippostrongylus brasiliensis infection induces jejunal mastocytosis associated with enteric nerve remodelling in rats. The aim of this study was to evaluate the intestinal motility responses to meals and to neurotransmitters involved in the control of gut motility (acetylcholine (carbachol), substance P and neurokinin A) in both control and N. brasiliensis‐infected rats 30 days post‐infection. All rats were equipped with NiCr electrodes in the jejunum to record myoelectrical activity. The duration of disruption of the jejunal migrating myoelectrical complexes (MMC) induced by the different stimuli was determined. Meal ingestion and substance P administration disrupted the MMC pattern for similar durations in the two groups. Carbachol and neurokinin A induced a significantly longer MMC disruption in post‐infected rats than in controls (125 ± 8.3 vs. 70 ± 6 min for carbachol 100 μg kg−1 and 51 ± 4 vs. 40 ± 2 for neurokinin A 50 μg kg−1). The enhanced motor response in postinfected rats was reduced by previous mast cell stabilization with ketotifen or mast cell degranulation with compound BrX 537 A. In conclusion, the increased intestinal motor reactivity to carbachol and neurokinin A in post‐N. brasiliensis‐infected rats depends upon intestinal mast cell hyperplasia and degranulation.

Enhanced intestinal motor response to cholecystokinin in post-Nippostrongylus brasiliensis-infected rats: modulation by CCK receptors and the vagus nerve

The jejunal inflammation induced in rats by the nematode Nippostrongylus brasiliensis is followed by intestinal neuroimmune alterations including mast cell hyperplasia and nerve remodelling. On the other hand, cholecystokinin (CCK) plays a pivotal role in the regulation of intestinal motility. The aim of this study was to determine whether the intestinal motor response to CCK is altered 30 days after infection by N. brasiliensis. Thus, CCK‐8 (50 μg kg–1 intraperitoneally) disrupted the pattern of jejunal migrating myoelectric complexes for a longer time in postinfected rats (95.5 ± 3.5 min) than in controls (48.1 ± 5.1 min). This enhanced jejunal response was also found after oral administration of the potent releaser of endogenous CCK, soybean trypsin inhibitor. In contrast, no alteration of the inhibition of colonic motility by CCK administration was observed. The increased responsiveness of jejunal motility to CCK persisted after mast cell stabilisation or depletion but was prevented by atropine, devazepide and L‐365260 (CCK‐A and CCK‐B receptor antagonists, respectively) and vagotomy. These results indicate that neuroimmune alterations after N. brasiliensis infection lead to an increased intestinal motility response to CCK that involves a cholinergic mediation, a vagal pathway and alterations in intestinal CCK‐A and CCK‐B receptors.

Involvement of tachykinin receptors in sensitisation to cow's milk proteins in guinea pigs

BACKGROUND There is growing evidence for a pivotal role for tachykinins in gut neuroimmune interactions. AIMS To determine whether NK1, NK2, and NK3 tachykinin receptors are involved in milk protein induced allergic sensitisation. METHODS Eight groups of 12 Dunkin-Hartley guinea pigs (250–300 g) were used. Four groups were sensitised to milk proteins for three weeks. During this period, these animals were injected intraperitoneally each day with NK1 (SR 140333; 0.3 mg/kg), NK2 (SR 48968; 5 mg/kg), or NK3 (SR 142801; 5 mg/kg) receptor antagonist or vehicle. The fifth group had water available instead of milk and was used as a non-sensitised control. The three other groups received the NK receptor antagonists for three weeks but were not sensitised to milk proteins. RESULTS Sensitised animals treated with NK1 and NK3 receptor antagonists had both lower IgE and IgG serum titres, evaluated by passive cutaneous anaphylaxis, and lower specific IgG serum titres, determined by enzyme linked immunosorbent assay (ELISA), than vehicle treated animals. Sensitisation induced an increase in intestinal mast cell number which was abolished by treatment with the NK1 receptor antagonist. Antigenic challenge-induced jejunal hypersecretion was also blocked by treatment with the NK1 receptor antagonist. CONCLUSION In guinea pigs, NK1 and NK3 but not NK2 receptors are involved in sensitisation to cow’s milk. However, NK1 but not NK3 receptor antagonists abolish both the hypermastocytosis induced by food allergy and the hypersecretion induced by antigenic challenge, suggesting different roles for NK1 and NK3 receptors in the mechanisms of sensitisation to β-lactoglobulin.

Functional evidence for NO‐synthase activation by substance P through a mechanism not involving classical tachykinin receptors in guinea‐pig ileum in vitro

1 This study tested the hypothesis that a nitric oxide synthase (NOS) was activated in guinea‐pig ileum in vitro in response to substance P (SP), and attempted to characterize the tachykinin receptor involved in this activation by the use of selective receptor agonists and antagonists. 2 Strips of guinea‐pig ileum (8 × 2 mm) were superfused (Krebs, 37°C, 2 ml min−1) with: (i) tachykinin receptor agonists: SP, GR 73,632 (NK1), GR 64,349 (NK2), senktide (NK3), and neuropeptide (NP)γ; (ii) tachykinin receptor antagonists: CP 99,994 (NK1), SR 48,968 (NK2), SR 142,801 (NK3); (iii) nerve‐related agents: carbachol (CCh), atropine, tetrodotoxin (TTX), hexamethonium; (iv) NOS inhibitors: N/>ω‐nitro‐L‐arginine‐methyl‐ester (L‐NAME), Nω‐monomethyl‐L‐arginine (L‐NMMA) and aminoguanidine (AG); (v) NO‐related agents, L‐arginine (L‐Arg), D‐arginine (D‐Arg), sodium nitroprusside (NaNP) and methaemoglobin. Muscle contractility was recorded isometrically and quantified as integrated area of activity. 3 SP, tachykinin receptor agonists and NPγ (10 pM to 10 μm), produced concentration‐dependent contractions of ileal strips, with EC50s in the nanomolar range, and maximal responses (Emax) attained at 0.1 μm for SP and 1 μm for the other agonists. The Emax response to SP equalled that to KCl (60 mM) taken as a 100% control (99.3% [93.0–105.7]; mean and 95% CI; n = 12); a comparable Emax contraction was obtained with the other tachykinin receptor agonists (1 μm) as well as with CCh (1 μm). 4 Under baseline conditions, L‐NAME (1 μm), L‐NMMA (1 μm) and AG (1 μm), failed to contract the muscle strip. In contrast, when superfused for 3 min, 10 min after SP (0.1 μm), they induced a transient contraction of the strip (e.g. for 1 μm L‐NAME: 50 to 70 s duration; amplitude 73 ± 12%, n = 24). 5 The NOS inhibitor‐induced contractile response was not obtained after KCl (60 mM), GR 73,632, GR 64,349, senktide or CCh (all up to 1 μm). In contrast, this contractile response was obtained after NPγ (1 μm). 6 Blockade of tachykinin NK1, NK2 and NK3 receptors by continuous superfusion of CP 99,994, SR 48,968 and SR 142,801 (1 μm) respectively, starting 5 min before SP, did not modify the response to L‐NAME, superfused 10 min after SP (0.1 μm). The contractile response to L‐NAME (1 μm) was blocked by atropine (1 μm), superfused either before or after SP. In contrast, it persisted after TTX or hexamethonium (1 μm) superfused in the same conditions. 7 The amplitude of NOS inhibitor‐induced contraction (1 μm) was dependent on the concentration of priming SP (1 pM to 1 μm). In contrast, the contractile response to NOS inhibitors (1 nM to 10 μm) of the ileum strip primed with SP (0.1 μm) was not concentration‐related. 8 L‐NAME‐induced contraction was prevented by continuous superfusion of L‐Arg (1 μm), but not D‐Arg (1 μm). In addition, the NO donor, sodium nitroprusside (1 μm) and the NO scavenger, methaemoglobin (10 μg ml−1), both prevented the contractile response to L‐NAME. 9 In summary, SP and to a lesser extent NPγ, exert a permissive action allowing contractile stimulating effects of L‐NAME, L‐NMMA and AG, in guinea‐pig ileum in vitro, by a mechanism which apparently does not involve tachykinin NK1, NK2 and NK3 receptors. This action is likely to result from the activation of a NO‐synthase by SP in the vicinity of intestinal myocytes. Thus, L‐NAME, L‐NMMA or AG, by blocking this SP‐induced NO production, unveiled a smooth muscle contraction which involves a cholinoceptor (atropine‐sensitive) mechanism.

Evaluation of the Effects of Sennosides on Uterine Motility in the Pregnant Ewe

The effects of sennosides on uterine motility were evaluated by electromyography in healthy adult ewes between day 70 and 120 of pregnancy to assess possible disturbances of the physiological pattern of contractility and eventual risks in pregnancy maintenance. At this stage of pregnancy, the ovine genital tract presented motility episodes of 6-8 min duration occurring at approximately hourly intervals. A dose-range study (10-160 mg/kg intracolonically) in 2 ewes showed that diarrhea was systematically obtained with doses greater than 20 mg/kg and was connected with a marked depression of both ileum and spiral colon motility. A standard dose of 60 mg/kg administered intracolonically 1-3 times at 7- to 10-day intervals to 12 ewes was used in the uterus studies. The experiments showed that sennosides did not stimulate uterine motility in the pregnant ewe, but slightly depressed it in some ewes. Cervix motility was never influenced. Intolerance of the drug was observed in half of the animals resulting mainly in anorexia or weakness and confirming a specific toxicity of senna in ruminants which is not known from other species. These effects were not related to uterine motility and pregnancy maintenance was normal in all ewes.