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.

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.

Neuropeptide S inhibits stress-stimulated faecal output in the rat.

Neuropeptide S (NPS) is a recently identified bioactive peptide that activates an orphan G-protein coupled receptor, called the NPS receptor (NPSR). In rats, NPS and NPSR constitute a novel neuropeptide system expressed both in the central nervous system and in peripheral tissues, controlling visceromotor, neuroendocrine, nociceptive and behavioural responses. To improve the knowledge of the role of the NPS-NPSR system in the gastrointestinal (GI) tract, we investigated: 1- the supraspinal effect of NPS on motor functions of the upper (gastric emptying and gastrointestinal transit) and lower (distal colonic transit and faecal output) GI tract under basal conditions, 2- during pathological states (restraint stress and corticotropin releasing factor (CRF)-induced defecation) in the rat, and 3- the receptor type involved in treatment with NPS using NPS, tachykinin NK(3) and opioid receptor antagonists (([D-Cys(tBu)5]NPS), SR142801 and naloxone, respectively). Intracerebroventricular injection of NPS failed to modify basal gastric emptying, gastrointestinal transit and distal colon propulsion, but significantly and dose-dependently reduced faecal pellet excretion and weight stimulated by restraint stress and CRF. The inhibitory effect of NPS on stress-induced defecation was unmodified by pre-treatment with either the tachykinin or opioid receptor antagonists, but was counteracted by a NPSR antagonist. The present study demonstrates, for the first time, that the supraspinal NPS system, which does not participate in the physiological control of GI motility, plays an inhibitory role on defecation stimulated by restraint stress and CRF. The combination of the ability of NPS to inhibit faecal output together with its known anxiolytic effect may be promising, especially in pathological conditions such as irritable bowel syndrome, where stress and the hyperactivity of the CRF system contribute to the co-morbidity of anxiety with colonic motor symptoms such as diarrhoea.

TLQP-21, a VGF-derived peptide, prevents ethanol-induced gastric lesions: insights into its mode of action.

BACKGROUND AND AIM: TLQP-21, a peptide derived from the vgf gene, has been reported to play a role in the regulation of rat gastric motility, but its influence on gastric mucosal integrity is unknown. EXPERIMENTAL APPROACH: We investigated the effects of central (0.8-8 nmol/rat) or peripheral (48-240 nmol/kg) TLQP-21 administration on ethanol- (EtOH, 50%, 1 ml/rat) induced gastric lesions in the rat. The mechanisms involved in such activity were also examined. RESULTS: Central TLQP-21 injection dose-dependently reduced EtOH-induced gastric lesions (ED(50) = 3.16 nmol), while peripheral TLQP-21 administration had no effect. The TLQP-21 gastroprotective effect against EtOH injury was accompanied by a significant increase in gastric prostaglandin E(2) (PGE(2)) production linked to an increase in constitutive cyclooxygenase (COX) expression. The nitric oxide (NO) synthase inhibitor L-NAME (70 mg/kg, s.c.), the nonselective COX inhibitor indomethacin (10 mg/kg, orally) and capsaicin denervation removed TLQP-21 gastroprotection. CONCLUSIONS: This study shows for the first time that central TLQP-21 exerts a protective action on the gastric mucosa exposed to the noxious agent EtOH. TLQP-21 gastroprotection is mediated by constitutive-derived NO and PGE(2), and requires the integrity of sensory nerve fibers.

The hypothalamus-pituitary-adrenal axis does not influence the protective effects of nociceptin/orphanin FQ on the rat gastric mucosa.

The participation of hypothalamus-pituitary-adrenal axis in the gastroprotective effects of nociceptin/orphanin FQ (N/OFQ) has been investigated. Gastric mucosal lesions were induced by intragastric administration of 50% ethanol, 1 ml/rat. Rats received N/OFQ either by the intracerebroventricular (icv) route, at 3 microg/rat, or by the intraperitoneal (ip) route, at 10 microg/kg, 30 min before ethanol administration. The protective effect of icv and ip administered N/OFQ was assessed in adrenalectomized rats and in rats pretreated with the glucocorticoid receptor antagonist, mifepristone, or with the CRF receptor antagonist, alpha-helical CRF(9-41). The damaging effect of ethanol was apparently not influenced by adrenalectomy. N/OFQ markedly reduced macroscopically and histologically assessed gastric mucosal damage. The extent of reduction by N/OFQ was comparable in adrenalectomized and in sham-operated rats, with either icv or ip route of administration. Pretreatment with mifepristone, both icv (80 microg/rat) and ip (10 mg/kg) injected, did not modify the response to icv and ip N/OFQ. Pretreatment with alpha-helical CRF(9-41) (25 microg/rat icv or 250 microg/kg ip), had no effect on the reduction of gastric damage produced by icv or ip N/OFQ. Present findings suggest that the gastroprotective effects of N/OFQ on ethanol-induced damage do not involve the endocrine pathway through the HPA axis.

Maternal exposure to low levels of corticosterone during lactation protects adult rat progeny against TNBS-induced colitis: A study on GR-mediated anti-inflammatory effect and prokineticin system.

The early phase of life represents a critical period for the development of an organism. Interestingly, early life experiences are able to influence the development of the gastrointestinal tract and the reactivity to colonic inflammatory stress. We recently demonstrated that adult male rats exposed to low doses of corticosterone during lactation (CORT-nursed rats) are protected against experimental colitis induced by the intracolonic infusion of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Based on these interesting results, we wanted to better investigate which cellular actors could be involved in the protection of CORT-nursed rats from TNBS-induced experimental colitis. Therefore, in the present work, we focused our attention on different factors implicated in GR-mediated anti-inflammatory effect. To address this issue, colonic tissues, collected from control and CORT-nursed healthy animals and from control and CORT-nursed colitic rats, were processed and the following inflammatory factors were evaluated: the expression of (i) glucocorticoid receptors (GR), (ii) glucocorticoid-induced leucine zipper (GILZ), (iii) phospho-p65NF-κB, (iv) the pro-inflammatory cytokines IL-1ß and TNF-α, (v) the prokineticins PK2 and PK2L and (vi) their receptors PKR1 and PKR2. We found that adult CORT-nursed rats, in comparison to controls, showed increased expression of colonic GR and reduced expression of pro-inflammatory molecules (IL-1ß, TNF-α, PK2 and PK2L) in response to inflammatory colitis. The observed changes were associated with an increase in GILZ colonic expression and with a reduction in phospo-p65NF-κB colonic expression.

Tachykinins: role in human gastrointestinal tract physiology and pathology.

Tachykinins (TKs) and their receptors (NK1, NK2 and NK3), which are diffusely expressed in the human gastrointestinal tract, represent an endogenous modulator system regulating enteric secretomotor functions, inflammatory and immune responses, and visceral hypersensitivity, mainly during pathological gut diseases. Pathophysiological implications of TKs in the digestive tract include changes in TK innervation, in the expression of TKs and TK receptors, which result in inflammation- and immune-induced disturbances of gut functions, such as dysmotility (diarrhoea/constipation), secretory diarrhoea and visceral hyperalgesia. Increasing evidence correlates all these TKergic system abnormalities with gastrointestinal diseases of different etiology (i.e. inflammatory bowel diseases, irritable bowel syndrome). Accordingly, TK receptors have been identified as novel targets for the development of new therapeutic agents for clinical use. Available preclinical findings have shown that TK antagonists could counteract the most significant symptoms characterizing these gut diseases.

Immunocytochemical distribution of NK-1 and NK-3 tachykinin receptors in isolated pancreatic acini of guinea pigs and rats.

In this study, we investigated the immunocytochemical distribution of NK-1 and NK-3 tachykinin receptors in guinea pig and rat isolated pancreatic acini. In dispersed acinar cells from guinea pig, immunofluorescence staining detected similar densities of NK-1 and NK-3 receptors; conversely, rat acinar cells expressed NK-1 receptors more strongly than NK-3 receptors. In line with previous functional studies, these immunocytochemical findings suggest that guinea pig NK-1 and NK-3 receptors and rat NK-1 receptors alone play a direct stimulatory role in the basal pancreatic acinar amylase release.

Gastrointestinal effects of intracerebroventricularly injected nociceptin/orphaninFQ in rats.

Nociceptin/orphanin FQ/(N/OFQ), a novel heptadecapeptide recently isolated from porcine and rat brain, is the endogenous ligand of the N/OFQ peptide receptor (NOP, previously known as ORL-1). In this study we examined the effects of intracerebroventricularly (icv) injected N/OFQ on gastric emptying, gastrointestinal transit, colonic propulsion and gastric acid secretion in rats. N/OFQ (0.01-10 nmol/rat) significantly delayed gastric emptying of a phenol red meal, inhibited transit of a non-absorbable charcoal marker through the small intestine and increased the mean colonic bead expulsion time. These N/OFQ-motor effects were abolished by the NOP receptor selective antagonist [NPhe(1)]N/OFQ(1-13)-NH(2) (50 nmol/rat), but were unaltered by the classical opioid receptor antagonist, naloxone (9.2 micromol/kg). Icv injected N/OFQ (10 nmol/rat) decreased gastric acid secretion in 2-h pylorus ligated rats in a naloxone sensitive manner. [NPhe(1)]N/OFQ(1-13)-NH(2) (100 nmol/rat) icv administered alone stimulated gastric acid secretion. These results indicate that N/OFQ activates via NOP receptor stimulation a central inhibitory pathway modulating gastrointestinal propulsive activity and gastric acid secretion in rats.

Central effects of selective NK1 and NK3 tachykinin receptor agonists on two models of experimentally-induced colitis in rats.

Peripheral tachykinins (TKs) are believed to play a role in the pathogenesis of inflammatory bowel diseases (IBD). In this study we investigated changes induced by central administration of two natural TK receptor agonists, NK(1) (PG-SPI) and NK(3) (PG-KII), on trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulphate (DSS)-induced experimental colitis in rats. Colitis was induced by instilling a single intracolonic dose of TNBS 50 mgkg(-1) (0.5 ml in 50% ethanol) or by oral administration of 5% DSS for 7 days. Each group of rats was intracerebroventricularly injected daily with PG-SPI and PG-KII (0.5, 5, and 50 microgkg(-1)). On day 3, TNBS-treated animals were killed and the severity of gut inflammation was evaluated by measuring myeloperoxidase (MPO) activity, interleukin-1beta (IL-1beta) production and by scoring macroscopic and histologic colonic damage. DSS-treated animals were checked daily for the length of survival and for stool consistency and faecal blood. In the TNBS group, PG-SPI and PG-KII increased scores for the severity of colonic damage, stimulated the production of IL-1beta and increased granulocyte infiltration into the colon (MPO activity). In the DSS group, PG-SPI and PG-KII decreased the percentage of surviving animals, and increased the number of rats that developed loose stools and blood in the faeces and the MPO activity. These results indicate that centrally injected NK(1) and NK(3) tachykinin receptor agonists play a proinflammatory role in experimentally-induced colitis in rats.

Maternal exposure to low levels of corticosterone during lactation protects against experimental inflammatory colitis-induced damage in adult rat offspring.

Opposing emotional events (negative/trauma or positive/maternal care) during the postnatal period may differentially influence vulnerability to the effects of stress later in life. The development and course of intestinal disorders such as inflammatory bowel disease are negatively affected by persistent stress, but to date the role of positive life events on these pathologies has been entirely unknown. In the present study, the effect of early life beneficial experiences in the development of intestinal dysfunctions, where inflammation and stress stimuli play a primary role, was investigated. As a "positive" experimental model we used adult male rat progeny nursed by mothers whose drinking water was supplemented with moderate doses of corticosterone (CORT) (0.2 mg/ml) during the lactation period. Such animals have been generally shown to cope better with different environmental situations during life. The susceptibility to inflammatory experimental colitis induced by intracolonic infusion of TNBS (2,4,6-trinitrobenzenesulphonic acid) was investigated in CORT-nursed rats in comparison with control rats. This mild increase in maternal corticosterone during lactation induced, in CORT-nursed rats, a long lasting protective effect on TNBS-colitis, characterized by improvements in some indices of the disease (increased colonic myeloperoxidase activity, loss of body weight and food intake) and by the involvement of endogenous peripheral pathways known to participate in intestinal disorder development (lower plasma corticosterone levels and colonic mast cell degranulation, alterations in the colonic expression of both corticotrophin releasing factor/CRF and its receptor/CRH-1R). All these findings contribute to suggesting that the reduced vulnerability to TNBS-colitis in CORT-nursed rats is due to recovery from the colonic mucosal barrier dysfunction. Such long lasting changes induced by mild hormonal manipulation during lactation, making the adult also better adapted to colonic inflammatory stress, constitute a useful experimental model to investigate the etiopathogenetic mechanisms and therapeutic treatments of some gastrointestinal diseases.

Peripheral anti-nociceptive effect of nociceptin/orphanin FQ in inflammation and stress-induced colonic hyperalgesia in rats.

Nociceptin/orphanin FQ (N/OFQ) and its NOP receptors are present in the central nervous system and in the periphery playing important roles in the modulation of gastrointestinal functions and pain. The aim of this study was to investigate the role of central and peripheral N/OFQ-NOP receptor system in the nociceptive response to colorectal distension (CRD) in basal condition and in two models of gut hypersensitivity triggered by both inflammation and stress. Male Wistar rats were tested in basal and in post-inflammatory conditions, i.e., 5 days after IC TNBS instillation (80 mg/Kg) and received N/OFQ (2 nmol/Kg IP), UFP-101 (a selective NOP receptor antagonist, 10 nmol/Kg IP), N/OFQ+UFP-101, N/OFQ (0.5 nmol/rat ICV) or vehicle. Female rats were tested in basal and after partial restraint stress receiving the same pharmacological treatment. CRD was performed using barostat and abdominal contractions were recorded by electromyography. In basal condition, N/OFQ, ICV and IP injected, did not modify basal visceral sensitivity. Both in TNBS and stress-induced hyperalgesia, IP but not ICV injection of N/OFQ significantly decreased the number of abdominal contractions. Peripheral injection of UFP-101 antagonized N/OFQ effect. Moreover, in post-inflammatory colitis, UFP-101, injected alone, exacerbated visceral hyperalgesia to CRD compared with vehicle. These findings indicate that in rats, N/OFQ, only peripherally injected, reduces visceral hypersensitivity triggered by inflammation or stress without affecting basal sensitivity. N/OFQ visceral anti-hyperalgesic effect involves peripheral NOP receptors. In a post-inflammatory, but not in an acute stress colitis model, N/OFQergic system is endogenously activated.

Regulation of pancreatic secretion in vitro by nociceptin/orphanin FQ and opioid receptors: a comparative study.

The effects of nociceptin/orphanin FQ (N/OFQ) on gastrointestinal functions resemble those of classic analgesic opioid agonists. In this study, we compared changes in amylase release from guinea pig isolated pancreatic acini and lobules induced by the N/OFQ analogue [Arg(14),Lys(15)]N/OFQ and by the delta-receptor opioid agonist deltorphin. Carbachol strongly stimulated amylase release from isolated acini. Both peptides left baseline and carbachol-stimulated amylase secretion from pancreatic acini unchanged. Co-incubation of KCl-stimulated lobules with [Arg(14),Lys(15)]N/OFQ or deltorphin inhibited KCl-induced amylase release in a concentration-dependent manner. Although maximal inhibition of amylase release by [Arg(14),Lys(15)]N/OFQ and deltorphin had similar amplitude, [Arg(14),Lys(15)]N/OFQ was 100-fold more potent than deltorphin on a molar basis. The selective NOP-receptor antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101) antagonized [Arg(14),Lys(15)]N/OFQ-induced inhibition but left deltorphin-induced inhibition unchanged. The selective delta opiate receptor antagonist naltrindole had no effect on [Arg(14),Lys(15)]N/OFQ inhibition but partly prevented the inhibition by deltorphin. [Arg(14),Lys(15)]N/OFQ and deltorphin combined had no influence on each other. These findings show that [Arg(14),Lys(15)]N/OFQ inhibits pancreatic enzyme secretion by suppressing cholinergic transmission in intralobular nerve fibers, as previously reported for opioid agents. They suggest that [Arg(14),Lys(15)]N/OFQ inhibition of amylase release is mediated through the NOP receptor and not through the delta opioid receptor. The N/OFQ-NOP receptor system, like the delta opioid system, plays an inhibitory role in regulating exocrine pancreatic secretion.

Expression of NK-1 and NK-3 tachykinin receptors in pancreatic acinar cells after acute experimental pancreatitis in rats.

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats.