seryl-leucyl-isoleucyl-glycyl--arginyl-leucinamide and Colitis

This study examined the contribution of mast cells to colon-bladder cross organ sensitization induced by colon irritation with trinitrobenzene sulfonic acid (TNBS-CI). In urethane anesthetized rats 12 days after TNBS-CI, the voiding interval was reduced from 357 s to 201 s and urothelial permeability, measured indirectly by absorption of sodium fluorescein from the bladder lumen, increased six-fold. These effects were blocked by oral administration of ketotifen (10 mg/kg, for 5 days), a mast cell stabilizing agent. TNBS-CI in wild type mice produced a similar decrease in voiding interval (from 319 s to 209 s) and a 10-fold increase in urothelial permeability; however this did not occur in KitªWª/KitªW-vª mast cell deficient mice. Contractile responses of bladder strips elicited by Compound 48/80 (50 μg/ml), a mast cell activating agent, were significantly larger in strips from rats with TNBS-CI (145% increase in baseline tension) than in control rats (55% increase). The contractions of strips from rats with TNBS-CI were reduced 80-90% by pretreatment of strips with ketotifen (20 μM), whereas contractions of strips from control animals were not significantly changed. Bladder strips were pretreated with SLIGRL-NH2 (100 μM) to desensitize PAR-2, the receptor for mast cell tryptase. SLIGRL-NH2 pretreatment reduced by 60-80% the 48/80 induced contractions in strips from rats with TNBS-CI but did not alter the contractions in strips from control rats. These data indicate that bladder mast cells contribute to the bladder dysfunction following colon-bladder cross-sensitization.

Topics: Animals; Colitis; Colon; Disease Models, Animal; Female; Ketotifen; Mast Cells; Membrane Transport Modulators; Mice; Mice, Knockout; Muscle Contraction; Neurons, Afferent; Oligopeptides; p-Methoxy-N-methylphenethylamine; Permeability; Proto-Oncogene Proteins c-kit; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Trinitrobenzenesulfonic Acid; Urinary Bladder; Urination Disorders

Many trypsin-like serine proteases such as β-tryptase are involved in the pathogenesis of colitis and inflammatory bowel diseases. Inhibitors of individual proteases show limited efficacy in treating such conditions, but also probably disrupt digestive and defensive functions of proteases. Here, we investigate whether masking their common target, protease-activated receptor 2 (PAR2), is an effective therapeutic strategy for treating acute and chronic experimental colitis in rats. A novel PAR2 antagonist (5-isoxazoyl-Cha-Ile-spiro[indene-1,4'-piperidine]; GB88) was evaluated for the blockade of intracellular calcium release in colonocytes and anti-inflammatory activity in acute (PAR2 agonist-induced) versus chronic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] models of colitis in Wistar rats. Disease progression (disease activity index, weight loss, and mortality) and postmortem colonic histopathology (inflammation, bowel wall thickness, and myeloperoxidase) were measured. PAR2 and tryptase colocalization were investigated by using immunohistochemistry. GB88 was a more potent antagonist of PAR2 activation in colonocytes than another reported compound, N¹-3-methylbutyryl-N⁴-6-aminohexanoyl-piperazine (ENMD-1068) (IC₅₀ 8 μM versus 5 mM). Acute colonic inflammation induced in rats by the PAR2 agonist SLIGRL-NH₂ was inhibited by oral administration of GB88 (10 mg/kg) with markedly reduced edema, mucin depletion, PAR2 receptor internalization, and mastocytosis. Chronic TNBS-induced colitis in rats was ameliorated by GB88 (10 mg/kg/day p.o.), which reduced mortality and pathology (including colon obstruction, ulceration, wall thickness, and myeloperoxidase release) more effectively than the clinically used drug sulfasalazine (100 mg/kg/day p.o.). These disease-modifying properties for the PAR2 antagonist in both acute and chronic experimental colitis strongly support a pathogenic role for PAR2 and PAR2-activating proteases and therapeutic potential for PAR2 antagonism in inflammatory diseases of the colon.

Topics: Animals; Body Weight; Calcium Signaling; Colitis; Colon; Cytokines; Edema; HT29 Cells; Humans; Indenes; Intestinal Mucosa; Male; Oligopeptides; Piperazines; Piperidines; Rats; Rats, Wistar; Receptor, PAR-2; Sulfasalazine; Survival Rate; Transendothelial and Transepithelial Migration; Trinitrobenzenesulfonic Acid; Tryptases; Ulcer

Protease-activated receptor (PAR)-2 plays important roles in intestinal inflammatory responses. Changes in PAR-2-mediated smooth muscle function may contribute pathophysiologically to the intestinal motility disorders often observed in inflammatory bowel disease (IBD). Stimulation of PAR-2 by trypsin-induced relaxation of carbachol- and KCl-induced contractions in normal rat colonic smooth muscle was completely resolved by tissue pretreatment with apamin, but not by pretreatment with l-NMMA or a cocktail of neuronal blockers (tetrodotoxin, hexamethonium and propranolol). In colon inflamed by dextran sodium sulphate (DSS), trypsin-induced inhibitory effects were significantly reduced. Relaxation induced by SLIGRL-NH(2), a selective PAR-2-activating peptide, was also reduced in DSS-treated rat colon. However, inhibitory effects of 1-ethylbenzimidazolin-2-one, an activator of small conductance Ca(2+)-activated K(+) channel, were unaffected. Expression of PAR-2 mRNA in colonic muscularis externa was significantly lower in DSS-treated rats than in control rats. These results suggest that the PAR-2 mediated relaxation system in colonic smooth muscle is suppressed in this experimental colitis rat model, and may contribute to motility disorders in IBD.

Topics: Animals; Apamin; Carbachol; Colitis; Colon; Dextran Sulfate; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Oligopeptides; Potassium Chloride; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Small-Conductance Calcium-Activated Potassium Channels; Trypsin

Proteinase-activated receptor-2 (PAR2) activation induces colonic inflammation by an unknown mechanism. We hypothesized that PAR2 agonists administered intracolonically in mice induce inflammation via a neurogenic mechanism. Pretreatment of mice with neurokinin-1 and calcitonin-gene-related peptide (CGRP) receptor antagonists or with capsaicin showed attenuated PAR2-agonist-induced colitis. Immunohistochemistry demonstrated a differential expression of a marker for the type-1 CGRP receptor during the time course of PAR2-agonist-induced colitis, further suggesting a role for CGRP. We conclude that PAR2-agonist-induced intestinal inflammation involves the release of neuropeptides, which by acting on their receptors cause inflammation. These results implicate PAR2 as an important mediator of intestinal neurogenic inflammation.

Topics: Animals; Calcitonin Gene-Related Peptide; Capsaicin; Colitis; Enteric Nervous System; Inflammation; Mice; Neurons, Afferent; Neuropeptides; Oligopeptides; Piperidines; Quinuclidines; Receptor, PAR-2; Receptors, Neurokinin-1

PAR-2s are highly expressed throughout the gastrointestinal tract. These receptors are cleaved by trypsin and mast cell tryptase and can be activated by peptides corresponding to the tethered ligand of the receptor (SLIGRL-NH2 for rat). The aim of this study was to determine whether colonic administration of PAR-2 agonists affects visceral sensitivity to rectal distention in conscious rats.. Abdominal contractions (a criteria of visceral pain) were recorded in rats equipped with intramuscular electrodes. Rectal distention was performed at various times after intracolonic infusion of SLIGRL-NH2 and trypsin. Inflammation parameters and permeability were followed in the colon after the intracolonic injections. Fos expression at a spinal level (L4-L6) was also studied 2 hours after intracolonic injection of SLIGRL-NH2.. Rectal distention significantly increased abdominal contractions starting at the RD volume of 0.8 mL. Intracolonic injection of SLIGRL-NH2 (200 microg/rat) and trypsin (200 U/rat), but not vehicle, LRGILS-NH2 (control peptide), boiled trypsin, or SLIGRL-NH2 injected IP, significantly increased (P < 0.05) abdominal contractions for high volumes of distention, 10- and 24-hour postinfusion. SLIGRL-NH2-induced hyperalgesia was inhibited by a NK1 receptor antagonist (SR 140333) but not by indomethacin. Intracolonic injection of SLIGRL-NH2 elevated spinal Fos expression and caused increased intestinal permeability but did not cause detectable inflammation.. Intracolonic infusion of subinflammatory doses of PAR-2 agonists activated spinal afferent neurons and produced a delayed rectal hyperalgesia that involves changes in intestinal permeability and the activation of NK1 receptors. These results identify a possible role for proteinases and PAR-2 in the genesis of visceral hyperalgesia.

Topics: Animals; Catheterization; Chelating Agents; Chromium Radioisotopes; Colitis; Colon; Compliance; Edetic Acid; Hyperalgesia; Injections, Intraperitoneal; Intestinal Absorption; Male; Neurons, Afferent; Oligopeptides; Prostaglandins; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Receptor, PAR-2; Receptors, Neurokinin-1; Receptors, Thrombin; Rectum; Signal Transduction; Spinal Cord; Substance P; Trypsin; Up-Regulation

The proteinase-activated receptor 2 (PAR-2) is a member of a family of G protein-coupled receptors for proteases. Proteases cleave PARs within the extracellular N-terminal domains to expose tethered ligands that bind to and activate the cleaved receptors. PAR-2 is highly expressed in colon in epithelial and neuronal elements. In this study we show that PAR-2 activation prevents the development and induces healing of T helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. A role for PAR-2 in the protection against colon inflammation was explored by the use of SLIGRL-NH(2), a synthetic peptide that corresponds to the mouse tethered ligand exposed after PAR-2 cleavage. TNBS-induced colitis was dose-dependently reduced by the administration of SLIGRL-NH(2), whereas the scramble control peptide, LSIGRL-NH(2), was uneffective. This beneficial effect was reflected by increased survival rates, improvement of macroscopic and histologic scores, decrease in mucosal content of T helper cell type 1 cytokines, protein, and mRNA, and a diminished myeloperoxidase activity. SLIGRL-NH(2), but not the scramble peptide, directly inhibited IFN-gamma secretion and CD44 expression on lamina propria T lymphocytes. Protection exerted by PAR-2 in TNBS-treated mice was reverted by injecting mice with a truncated form of calcitonin gene-related peptide and by sensory neurons ablation with the neurotoxin capsaicin. Collectively, these studies show that PAR-2 is an anti-inflammatory receptor in the colon and suggest that PAR-2 ligands might be effective in the treatment of inflammatory bowel diseases.

Topics: Animals; Calcitonin Gene-Related Peptide; Capsaicin; Cells, Cultured; Colitis; Colon; Disease Models, Animal; Down-Regulation; Enzyme Activation; Hyaluronan Receptors; Interferon-gamma; Interleukin-12; Interleukin-2; Mice; Mice, Inbred BALB C; Oligopeptides; Peptide Fragments; Receptor, PAR-2; Receptors, Thrombin; T-Lymphocytes; Trinitrobenzenesulfonic Acid