Protease-activated receptor 2 contributes to Toxoplasma gondii-mediated gut inflammation.

Toxoplasma gondii is a widespread intracellular parasite, which naturally enters the organism via the oral route and crosses the intestinal barrier to disseminate. In addition to neuronal and ocular pathologies, this pathogen also causes gut inflammation in a number of animals. This infection-triggered inflammation has been extensively studied in the C57BL/6 mice, highlighting the importance of the immune cells and their mediators in the development of gut pathology. However, despite their importance in inflammation, the role of protease-activated receptors (PAR) was never reported in the context of T.gondii-mediated small intestine inflammation. Using genetically modified mice, we show that PAR2 plays a pathogenic role in the development of gut inflammatory lesions. We find that PAR2 controls the innate inflammatory mediators IL-6, KC/CXCL1, PGE2 as well as neutrophil infiltration in T. gondii-triggered gut damage. These results bring new knowledge on the mechanisms operating in the gut in response to T. gondii infection.

Targeting fatty acid amide hydrolase and transient receptor potential vanilloid-1 simultaneously to modulate colonic motility and visceral sensation in the mouse: A pharmacological intervention with N-arachidonoyl-serotonin (AA-5-HT).

BACKGROUND: Endocannabinoid anandamide (AEA) inhibits intestinal motility and visceral pain, but it may also be proalgesic through transient receptor potential vanilloid-1 (TRPV1). AEA is degraded by fatty acid amide hydrolase (FAAH). This study explored whether dual inhibition of FAAH and TRPV1 reduces diarrhea and abdominal pain. METHODS: Immunostaining was performed on myenteric plexus of the mouse colon. The effects of the dual FAAH/TRPV1 inhibitor AA-5-HT on electrically induced contractility, excitatory junction potential (EJP) and fast (f) and slow (s) inhibitory junction potentials (IJP) in the mouse colon, colonic propulsion and visceromotor response (VMR) to rectal distension were studied. The colonic levels of endocannabinoids and fatty acid amides were measured. KEY RESULTS: CB1-positive neurons exhibited TRPV1; only some TRPV1 positive neurons did not express CB1. CB1 and FAAH did not colocalize. AA-5-HT (100 nM-10 µM) decreased colonic contractility by ~60%; this effect was abolished by TRPV1 antagonist 5'-IRTX, but not by CB1 antagonist, SR141716. AA-5-HT (1 µM-10 µM) inhibited EJP by ~30% and IJPs by ~50%. The effects of AA-5-HT on junction potentials were reversed by SR141716 and 5`-IRTX. AA-5-HT (20 mg/kg; i.p.) inhibited colonic propulsion by ~30%; SR141716 but not 5`-IRTX reversed this effect. AA-5-HT decreased VMR by ~50%-60%; these effects were not blocked by SR141716 or 5`-IRTX. AA-5-HT increased AEA in the colon. CONCLUSIONS AND INFERENCES: The effects of AA-5-HT on visceral sensation and colonic motility are differentially mediated by CB1, TRPV1 and non-CB1/TRPV1 mechanisms, possibly reflecting the distinct neuromodulatory roles of endocannabinoid and endovanilloid FAAH substrates in the mouse intestine.

A novel orally administered trimebutine compound (GIC-1001) is anti-nociceptive and features peripheral opioid agonistic activity and Hydrogen Sulphide-releasing capacity in mice.

BACKGROUND: Trimebutine maleate, a noncompetitive spasmolytic agent with some affinity for peripheral µ- and κ-opioid receptors has been evaluated as a treatment in a limited number of patients undergoing sedation-free full colonoscopy. The efficiency of such treatment was comparable to sedation-based colonoscopies to relieve from pain and discomfort. METHODS: A new and improved trimebutine salt capable of releasing in vivo hydrogen sulphide (H2S), a gaseous mediator known to reduce nociception, has been developed. This drug salt (GIC-1001) is composed of trimebutine bearing a H2S-releasing counterion (3-thiocarbamoylbenzoate, 3TCB), the latter having the ability to release H2S. GIC-1001 has been tested here in a mouse model of colorectal distension. RESULTS: In mice, while orally given trimebutine (the maleate salt, non-H2 S-releaser) only slightly reduced the nociceptive response to increasing pressures of colorectal distension, oral administration of GIC-1001 (the H2S-releaser) was able to significantly reduce nociceptive response to all noxious stimuli, in a dose-dependent manner. This effect of GIC-1001 was significantly better than the effects of its parent compound trimebutine administered at equimolar doses. CONCLUSIONS: Taken together, these results demonstrated increased antinociceptive properties for GIC-1001 compared to trimebutine, suggesting that this compound would be a better option to relieve from visceral pain and discomfort induced by lumenal distension.

Protective effects of n-6 fatty acids-enriched diet on intestinal ischaemia/reperfusion injury involve lipoxin A4 and its receptor.

BACKGROUND AND PURPOSE: Long-term intake of dietary fatty acids is known to predispose to chronic inflammation, but their effects on acute intestinal ischaemia/reperfusion (I/R) injury is unknown. The aim of this study was to determine the consequences of a diet rich in n-3 or n-6 polyunsaturated fatty acids (PUFA) on intestinal I/R-induced damage. EXPERIMENTAL APPROACH: Mice were fed three different isocaloric diets: a balanced diet used as a control and two different PUFA-enriched diets, providing either high levels of n-3 or of n-6 PUFA. Intestinal injury was evaluated after intestinal I/R. PUFA metabolites were quantitated in intestinal tissues by LC-MS/MS. KEY RESULTS: In control diet-fed mice, intestinal I/R caused inflammation and increased COX and lipoxygenase-derived metabolites compared with sham-operated animals. Lipoxin A4 (LxA4 ) was significantly and selectively increased after ischaemia. Animals fed a high n-3 diet did not display a different inflammatory profile following intestinal I/R compared with control diet-fed animals. In contrast, intestinal inflammation was decreased in the I/R group fed with high n-6 diet and level of LxA4 was increased post-ischaemia compared with control diet-fed mice. Blockade of the LxA4 receptor (Fpr2), prevented the anti-inflammatory effects associated with the n-6 rich diet. CONCLUSIONS AND IMPLICATIONS: This study indicates that high levels of dietary n-6, but not n-3, PUFAs provides significant protection against intestinal I/R-induced damage and demonstrates that the endogenous production of LxA4 can be influenced by diet.

Activation of the endogenous nociceptin system by selective nociceptin receptor agonist SCH 221510 produces antitransit and antinociceptive effect: a novel strategy for treatment of diarrhea-predominant IBS.

BACKGROUND: Diarrhea-predominant irritable bowel syndrome (IBS-D) is a functional gastrointestinal (GI) disorder, defined by the presence of loose stools and abdominal pain. In search for a novel anti-IBS-D therapy, here we investigated the nociceptin receptor (NOP)-dependent effects in the GI tract. METHODS: A novel potent and selective NOP agonist SCH 221510 was used in the study. The effect of NOP activation on mouse intestinal motility was characterized in vitro and in vivo, in physiological conditions and in animal models of hypermotility and diarrhea. Well-established mouse models of visceral pain were used to characterize the antinociceptive effect of the NOP activation. To provide additional evidence that the endogenous nociceptin system is a relevant target for IBS, NOP expression and nociceptin levels were quantified in serum and colonic biopsies from IBS-D patients. KEY RESULTS: SCH 221510 produced a potent NOP-mediated inhibitory effect on mouse intestinal motility in vitro and in vivo in physiological conditions. The NOP agonist displayed an antidiarrheal and analgesic action after oral administration in animal models mimicking the symptoms of IBS-D. Studies on human samples revealed a strong decrease in endogenous nociceptin system expression in IBS-D patients compared with healthy controls. CONCLUSIONS & INFERENCES: Collectively, mouse and human data suggest that the endogenous nociceptin system is involved in IBS-D and may become a target for anti-IBS-D treatments using potent and selective synthetic NOP agonists.

Chronic stress mediators act synergistically on colonic nociceptive mouse dorsal root ganglia neurons to increase excitability.

BACKGROUND: Stress hormones can signal to colonic dorsal root ganglia (DRG) neurons and may play a role in sustained hyperexcitability of nociceptors. METHODS: Mouse DRG neurons were exposed overnight to epinephrine (Epi) 5 nM and/or corticosterone (Cort) 1 µM or prior water-avoidance stress. Patch clamp recordings, visceromotor reflexes (VMRs) and molecular studies were conducted. KEY RESULTS: Water-avoidance stress induced neuronal hyperexcitability. Incubation of DRG neurons in both Cort and Epi (but neither alone) induced hyperexcitability (rheobase decreased 51%, p < 0.05; action potential discharge increased 95%, p < 0.01); this was blocked by antagonists of the ß2 adrenoreceptor (butoxamine, But) and Cort receptor (mifepristone) in combination or alone. Stress hormones enhanced voltage-gated Nav 1.7 currents (p < 0.05) and suppressed IA (p < 0.0001) and IK+ (p < 0.05) currents. Furthermore, stress hormones increased DRG ß2 adrenoreceptor mRNA (59%, p = 0.007) and protein (125%, p < 0.05), also Nav 1.7 transcript (45%, p = 0.004) and protein (114%, p = 0.002). In whole-animal studies, the WAS hyperexcitability of DRG neurons was blocked by antagonists of the ß2 and glucocorticoid receptors (GCR) but together they paradoxically increased VMRs to colorectal balloon distension. CONCLUSIONS & INFERENCES: Stress mediators Epi and Cort activate ß2 and GCR on DRG neurons which synergistically induce hyperexcitability of nociceptive DRG neurons and cause corresponding changes in voltage-gated Na(+) and K(+) currents. Furthermore, they increase the expression of ß2 adrenoreceptors and Nav1.7 channels, suggesting transcriptional changes could contribute to sustained signaling following stress. The paradoxical effects of But and mifepristone in electrophysiological compared to VMR testing may reflect different peripheral and central actions on sensory signaling.

Inhibition of sensory afferents activation and visceral pain by a brominated algal diterpene.

BACKGROUND: In the search of new therapeutic options for the treatment of pain, isolation, and testing of secondary metabolites from plant extracts has raised significant attention. We have investigated the effects of the brominated diterpene O(11) 15- cyclo-14-bromo-14,15-dihydrorogiol-3,11-diol (that we have named VLC5), extracted from the Mediterranean red algae Laurencia glandulifera. METHODS: The pure extract was tested on primary afferent calcium signals induced by high concentration of KCl, transcient receptor potential vanilloid (TRPV)1 (capsaicin) or TRPV4 agonists, histamine, or protease-activated receptor-2 (PAR(2) ) agonist. It was also tested in mice in a model of mustard oil-induced colonic hypersensitivity. KEY RESULTS: VLC5 was inhibited PAR(2) agonist or histamine-induced calcium mobilization in mouse primary afferents, but did not modify calcium signals induced by high concentrations of KCl, TRPV1 or TRPV4 agonists. The effect of VLC5 on histamine-induced calcium signal in primary afferent was inhibited by pertussis toxin pretreatment and was dependent on the activation of mu- or kappa-opioid receptor agonists, as it was inhibited by selective antagonists of those two receptors, but not by selective antagonist of the delta-opioid receptor. Intraperitoneal treatment of mice with VLC5 (10 mg kg(-1)) significantly reduced visceral pain behaviors induced by the intracolonic administration of mustard oil, in an opioid receptor-dependent manner. CONCLUSIONS & INFERENCES: We have demonstrated significant analgesic properties for the algal metabolite VLC5, which is able to signal directly to primary afferents, through a mechanism dependent on the activation of opioid receptors. This identifies a new natural compound capable of activating peripheral opioidergic systems, exerting analgesic properties.

PAR(2) and temporomandibular joint inflammation in the rat.

The proteinase-activated receptor 2 (PAR(2)) is a putative therapeutic target for arthritis. We hypothesized that the early pro-inflammatory effects secondary to its activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR(2) in retrogradely labeled trigeminal ganglion neurons. Furthermore, PAR(2) immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance-P-containing peripheral sensory nerve fibers. The intra-articular injection of PAR(2) agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx, and induction of mechanical allodynia. The pharmacological blockade of natural killer 1 (NK(1)) receptors abolished PAR(2)-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR(2) activation is pro-inflammatory in the TMJ, through a neurogenic mechanism involving NK(1) receptors. This suggests that PAR(2) is an important component of innate neuro-immune response in the rat TMJ.

A role for transient receptor potential vanilloid 4 in tonicity-induced neurogenic inflammation.

BACKGROUND AND PURPOSE: Changes in extracellular fluid osmolarity, which occur after tissue damage and disease, cause inflammation and maintain chronic inflammatory states by unknown mechanisms. Here, we investigated whether the osmosensitive channel, transient receptor potential vanilloid 4 (TRPV4), mediates inflammation to hypotonic stimuli by a neurogenic mechanism. EXPERIMENTAL APPROACH: TRPV4 was localized in dorsal root ganglia (DRG) by immunofluorescence. The effects of TRPV4 agonists on release of pro-inflammatory neuropeptides from peripheral tissues and on inflammation were examined. KEY RESULTS: Immunoreactive TRPV4 was detected in DRG neurones innervating the mouse hindpaw, where it was co-expressed in some neurones with CGRP and substance P, mediators of neurogenic inflammation. Hypotonic solutions and 4alpha-phorbol 12,13-didecanoate, which activate TRPV4, stimulated neuropeptide release in urinary bladder and airways, sites of neurogenic inflammation. Intraplantar injection of hypotonic solutions and 4alpha-phorbol 12,13-didecanoate caused oedema and granulocyte recruitment. These effects were inhibited by a desensitizing dose of the neurotoxin capsaicin, antagonists of CGRP and substance P receptors, and TRPV4 gene knockdown or deletion. In contrast, antagonism of neuropeptide receptors and disruption of TRPV4 did not prevent this oedema. TRPV4 gene knockdown or deletion also markedly reduced oedema and granulocyte infiltration induced by intraplantar injection of formalin. CONCLUSIONS AND IMPLICATIONS: Activation of TRPV4 stimulates neuropeptide release from afferent nerves and induces neurogenic inflammation. This mechanism may mediate the generation and maintenance of inflammation after injury and during diseases, in which there are changes in extracellular osmolarity. Antagonism of TRPV4 may offer a therapeutic approach for inflammatory hyperalgesia and chronic inflammation.

Protease-activated receptor-4 (PAR 4): a role as inhibitor of visceral pain and hypersensitivity.

Protease-activated receptor-4 (PAR(4)) belongs to the family of receptors activated by the proteolytic cleavage of their extracellular N-terminal domain and the subsequent binding of the newly released N-terminus. While largely expressed in the colon, the role of PAR(4) in gut functions has not been defined. We have investigated the effects of PAR(4) agonist on colonic sensations and sensory neuron signalling, and its role in visceral pain. We observed that a single administration of the PAR(4) agonist peptide (AYPGKF-NH(2)), but not the control peptide (YAPGKF-NH(2)) into the colon lumen of mice significantly reduced the visceromotor response to colorectal distension at different pressures of distension. Further, intracolonic administration of the PAR(4) agonist, but not the control peptide, was able to significantly inhibit PAR(2) agonist- and transcient receptor potential vanilloid-4 (TRPV4) agonist-induced allodynia and hyperalgesia in response to colorectal distension. Protease-activated receptor-4 was detected in sensory neurons projecting from the colon, and isolated from the dorsal root ganglia, where it co-expressed with PAR(2) and TRPV4. In total sensory neurons, PAR(4) agonist exposure inhibited free intracellular calcium mobilization induced by the pro-nociceptive agonists of PAR(2) and TRPV4. Finally, PAR(4)-deficient mice experienced increased pain behaviour in response to intracolonic administration of mustard oil, compared with wild-type littermates. These results show that PAR(4) agonists modulate colonic nociceptive response, inhibit colonic hypersensitivity and primary afferent responses to pro-nociceptive mediators. Endogenous activation of PAR(4) also plays a major role in controlling visceral pain. These results identify PAR(4) as a previously unknown modulator of visceral nociception.

Protease-activated receptor-4: a novel mechanism of inflammatory pain modulation.

BACKGROUND AND PURPOSE: Protease-activated receptor-4 (PAR(4)), the most recently discovered member of the PARs family, is activated by thrombin, trypsin and cathepsin G, but can also be selectively activated by small synthetic peptides (PAR(4)-activating peptide, PAR(4)-AP). PAR(4) is considered a potent mediator of platelet activation and inflammation. As both PAR(1) and PAR(2) have been implicated in the modulation of nociceptive mechanisms, we investigated the expression of PAR(4) in sensory neurons and the effects of its selective activation on nociception. EXPERIMENTAL APPROACH AND KEY RESULTS: We demonstrated the expression of PAR(4) in sensory neurons isolated from rat dorsal root ganglia by reverse transcription-polymerase chain reaction and immunofluorescence. We found that PAR(4) colocalized with calcitonin gene-related peptide and substance P. We also showed that a selective PAR(4)-AP was able to inhibit calcium mobilization evoked by KCl and capsaicin in rat sensory neurons. Moreover, the intraplantar injection of a PAR(4)-AP significantly increased nociceptive threshold in response to thermal and mechanical noxious stimuli, while a PAR(4) inactive control peptide had no effect. The anti-nociceptive effects of the PAR(4)-AP were dose-dependent and occurred at doses below the threshold needed to cause inflammation. Finally, co-injection of the PAR(4)-AP with carrageenan significantly reduced the carrageenan-induced inflammatory hyperalgesia and allodynia, but had no effect on inflammatory parameters such as oedema and granulocyte infiltration. CONCLUSIONS AND IMPLICATIONS: Taken together, these results identified PAR(4) as a novel potential endogenous analgesic factor, which can modulate nociceptive responses in normal and inflammatory conditions.

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

Luminal activation of protease-activated receptors-2 (PAR(2)) on colonocytes by trypsin or PAR(2)-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 PAR(2) expression in mice. CPP was assessed with (51)Cr-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 PAR(2) and assessment of effects of PAR(2) 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 PAR(2) expression, but with no change in CPP and MMCP-1 content. Trypsin administration restored PAR(2) 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 PAR(2).

The C-terminus of murine S100A9 protein inhibits hyperalgesia induced by the agonist peptide of protease-activated receptor 2 (PAR2).

BACKGROUND AND PURPOSE: S100A9 protein induces anti-nociception in rodents, in different experimental models of inflammatory pain. Herein, we investigated the effects of a fragment of the C-terminus of S100A9 (mS100A9p), on the hyperalgesia induced by serine proteases, through the activation of protease-activated receptor-2 (PAR2). EXPERIMENTAL APPROACH: Mechanical and thermal hyperalgesia induced by PAR2 agonists (SLIGRL-NH2 and trypsin) was measured in rats submitted to the paw pressure or plantar tests, and Egr-1 expression was determined by immunohistochemistry in rat spinal cord dorsal horn. Calcium flux in human embryonic kidney cells (HEK), which naturally express PAR2, in Kirsten virus-transformed kidney cells, transfected (KNRK-PAR2) or not (KNRK) with PAR2, and in mouse dorsal root ganglia neurons (DRG) was measured by fluorimetric methods. KEY RESULTS: mS100A9p inhibited mechanical hyperalgesia induced by trypsin, without modifying its enzymatic activity. Mechanical and thermal hyperalgesia induced by SLIGRL-NH2 were inhibited by mS100A9p. SLIGRL-NH2 enhanced Egr-1 expression, a marker of nociceptor activation, and this effect was inhibited by concomitant treatment with mS100A9p. mS100A9p inhibited calcium mobilization in DRG neurons in response to the PAR2 agonists trypsin and SLIGRL-NH2, but also in response to capsaicin and bradykinin, suggesting a direct effect of mS100A9 on sensory neurons. No effect on the calcium flux induced by trypsin or SLIGRL in HEK cells or KNRK-PAR2 cells was observed. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that mS100A9p interferes with mechanisms involved in nociception and hyperalgesia and modulates, possibly directly on sensory neurons, the PAR2-induced nociceptive signal.

The C-terminus of murine S100A9 protein inhibits hyperalgesia induced by the agonist peptide of protease-activated receptor 2 (PAR2)

Background and purpose: S100A9 protein induces anti-nociception in rodents, in different experimental models of inflammatory pain. Herein, we investigated the effects of a fragment of the C-terminus of S100A9 (mS100A9p), on the hyperalgesia induced by serine proteases, through the activation of protease-activated receptor-2 (PAR2). Experimental approach: Mechanical and thermal hyperalgesia induced by PAR2 agonists (SLIGRL-NH2 and trypsin) was measured in rats submitted to the paw pressure or plantar tests, and Egr-1 expression was determined by immunohistochemistry in rat spinal cord dorsal horn. Calcium flux in human embryonic kidney cells (HEK), which naturally express PAR2, in Kirsten virus-transformed kidney cells, transfected (KNRK-PAR2) or not (KNRK) with PAR2, and in mouse dorsal root ganglia neurons (DRG) was measured by fluorimetric methods. Key results: mS100A9p inhibited mechanical hyperalgesia induced by trypsin, without modifying its enzymatic activity. Mechanical and thermal hyperalgesia induced by SLIGRL-NH2 were inhibited by mS100A9p. SLIGRL-NH2 enhanced Egr-1 expression, a marker of nociceptor activation, and this effect was inhibited by concomitant treatment with mS100A9p. mS100A9p inhibited calcium mobilization in DRG neurons in response to the PAR2 agonists trypsin and SLIGRL-NH2, but also in response to capsaicin and bradykinin, suggesting a direct effect of mS100A9 on sensory neurons. No effect on the calcium flux induced by trypsin or SLIGRL in HEK cells or KNRK-PAR2 cells was observed. Conclusions and implications: These data demonstrate that mS100A9p interferes with mechanisms involved in nociception and hyperalgesia and modulates, possibly directly on sensory neurons, the PAR2-induced nociceptive signal.

Proteases and protease-activated receptors (PARs): novel signals for pain.

The recent detection of protease-activated receptors (PARs) on neurons of the peripheral and central nervous systems suggests that PARs and proteases that activate them, might be involved in neuronal functions. Among those functions, a particular focus on nociception has attracted considerable interest. The present article summarizes recent research progress on proteases and PARs as nociceptive signaling molecules in the nervous system and presents them as exciting new targets for therapeutic intervention in pain.