Protease‐Activated Receptor 2 Activation Provokes an Increase in Intracellular Calcium and Serotonin Secretion in a Human Enteroendocrine Cell Line

Studies have indicated that nearly half of all surgical patients still have inadequate pain relief. Thus, it is crucial to understand the mechanisms involved in postoperative pain in order to better treat it. Thus, the aim of this study was to investigate the involvement of mast cell degranulation, tryptase and its substrate, the protease-activated receptor 2, in a model of postoperative pain in mice. We evaluated the effect of the compound 48/80 (to cause mast cell mediator depletion), cromoglycate or ketotifen (mast cell stabilizers), gabexate (tryptase inhibitor) or N3-methylbutyryl-N-6-aminohexanoyl-piperazine (protease-activated receptor 2 antagonist) in a postoperative pain model in mice (n = 5-10). Mast cell degranulation and tryptase activity were also evaluated in the operated tissue (n= 5-8). The pre-treatment with compound 48/80 or ketotifen was able to prevent nociception throughout the postoperative hyperalgesia course (until 5 days after surgery), whereas cromoglycate presented a shorter effect (until 1 day). Gabexate or N3-methylbutyryl-N-6-aminohexanoyl-piperazine also produced a short-lasting effect in preventing postoperative nociception. However, neither gabexate, N3-methylbutyryl-N-6-aminohexanoyl-piperazine nor cromoglycate was capable of reversing nociception when administered after incision. Surgery led to early mast cell degranulation on the incised tissue and increased tryptase activity in tissue perfusates. Cromoglycate fully prevented the tryptase release in the perfusate and the compound 48/80 substantially reduced tryptase activity in the incised tissue. Thus, the mast cell degranulation with the subsequent release of tryptase and protease-activated receptor 2 activation are potential targets for the development of novel therapies to prevent, but not reverse, postoperative pain.

Functions and Imaging of Mast Cell and Neural Axis of the Gut

As a prerequisite for serotonin secretion, the P‐STS ileal enterochromaffin cell line responds to acetylcholine (ACh) stimulation with an increase in intracellular calcium mediated by the muscarinic ACh receptor M3 (M3R). Histamine increases intracellular calcium via histamine H1 receptor (H1R) in P‐STS cells and pre‐incubation with histamine specifically augments the response to ACh but not to epinephrine or nicotine. We aimed to elucidate whether histamine receptors are involved in this synergism. Astonishingly, HEK‐293 T cells—known to express M3R, but only a very low amount of histamine receptor messenger RNA—showed a similar enhancement of the calcium response to ACh by pre‐incubation with histamine. Despite the much lower level of H1R protein detected in HEK‐293 T cells as compared to P‐STS cells, in both cell lines pre‐treatment with H1R antagonists inhibited the synergism between histamine and ACh. No indication for an involvement of histamine H2 or H4 receptors in the synergism was found. Furthermore, pre‐incubation with the cAMP‐inducing compound forskolin had no influence on the intracellular calcium response to ACh. Serotonin secretion from P‐STS cells was increased after challenge with ACh and histamine added simultaneously compared to ACh alone, suggesting that histamine increases ACh‐induced serotonin secretion from enterochromaffin cells. In conclusion, our data suggest that histamine enhances the M3R‐mediated intracellular calcium response to ACh via activation of H1R. This probably increases serotonin secretion from enterochromaffin cells and thereby affects intestinal motility in histamine intolerance, food allergies and irritable bowel syndrome.

Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis

Growing evidence suggests that protease activated receptors (PARs) are mediators of persistent neuropathic pain, but their possible function as mediators in patients with post infectious irritable bowel syndrome (PI-IBS) remains to be further explored. This article aims to investigate the expression of PAR(2) and PAR(4) in the colonic mucosa of patients with PI-IBS, focusing on correlation with mast cell activation status. A total of 17 normal controls and 23 patients with PI-IBS volunteered the study. The expression and localization of PAR(2) and PAR(4) were investigated by RT-PCR and immunohistochemistry, and the expression of PAR(2) and PAR(4) in the mast cells was examined using double-immunofluorescence staining. The immunohistochemical study revealed that epithelial and submucosal cells showed immunoreactivity for both PAR(2) and PAR(4). Protease activated receptor 4 mRNA expression and immunoreactivity were down-regulated in PI-IBS compared with the control group. Specifically, a reduced immunoreactivity for PAR(4) was observed in mast cells of PI-IBS compared with normal controls, whereas there are no significant differences shown in PAR(2) between the PI-IBS and the control group. It is also found that the PAR(4) immunoreactivity decreases, while the activity of mast cells increases in PI-IBS rather than normal controls. This study outlines the down-regulation of PAR(4) in the mast cells of PI-IBS. It could be of considerable interests in understanding the mechanisms involved in the persistent colonic hypersensitivity and their potential role as therapeutic targets for PI-IBS.

Background : Protease activated receptor 2 (PAR2) is a member of the seven transmembrane G-protein coupled receptor (GPCR) family. Activation of PAR2 is cardioprotective in ex vivo and in vivo myocardial ischemia reperfusion model, potentially by cognate ligands released at reperfusion (R). Postconditioning (postcon), defined as alternating brief (seconds) episodes of R and ischemia applied at the onset of R , is also cardioprotective. This cardioprotection involves activation of GPCR by adenosine, bradykinin and opioids. However, the role of the GPCR PAR2 in cardioprotection by postcon has not been investigated. Hypothesis : This study tested the hypothesis that cardioprotection by postcon is mediated in part by endogenous PAR2 activation. Methods : Rats were randomly assigned to one of 5 groups with 30 min left coronary artery (LCA) occlusion followed by 3 h reperfusion: Control: no intervention was applied either before or after LCA occlusion (n =8); Postcon alone: 3 cycles of 10-s full reperfusion and 10-s re-occlusion were initiated at the onset of R (n=8); PAR2 antagonist alone: the selective PAR2 antagonist (FSLLRY-NH2, 1 mg/Kg) was injected 5 min before R (n=8); PAR2 antagonist + Postcon: PAR2 antagonist (1 mg/Kg) was administered 5 min before Postcon (n=8); Delayed PAR2 antagonist: PAR2 antagonist (1 mg/Kg) was given 5 min after the postcon protocol (n=8). Results: Area at risk (AAR) was comparable in all groups (35–38%). Compared to control, infarct size (TTC, area of necrosis/AAR, %) was significantly reduced by postcon alone (39.0% ± 1.3% vs 53.7% ± 1.5%, P<0.05). The PAR2 antagonist alone administered just before R had no effect on infarct size (57.4% ± 2.4% vs 53.7% ± 1.5%). Interestingly, the infarct sparing effect of postcon was completely reversed by PAR2 antagonist administered before postcon to control values ( 56.7% ± 2.7% *). Furthermore, the infarct sparing effect of postcon was also blocked by PAR2 antagonist given after postcon (50.3% ± 3.3% *). These results suggest that endogenous PAR2 activation induced by postcon during the early moments of R is cardioprotective. Conclusions : Cardioprotection by postcon is mediated, in part, by activation of endogenous PAR2, and suggests a general activation of GPCR. *P<0.05 vs Postcon.

Intervertebral disk (IVD) degeneration affects both humans and canines and is a major cause of low back pain (LBP). Mast cell (MC) and macrophage (MØ) infiltration has been identified in the pathogenesis of IVD degeneration (IVDD) in the human and rodent model but remains understudied in the canine. MC degranulation in the IVD leads to a pro-inflammatory cascade and activates protease activated receptor 2 (PAR2) on IVD cells. The objectives of the present study are to: (1) highlight the pathophysiological changes observed in the degenerate canine IVD, (2) further characterize the inflammatory effect of MCs co-cultured with canine nucleus pulposus (NP) cells, (3) evaluate the effect of construct stiffness on NP and MCs, and (4) identify potential therapeutics to mitigate pathologic changes in the IVD microenvironment. Canine IVD tissue was isolated from healthy autopsy research dogs (beagle) and pet dogs undergoing laminectomy for IVD herniation. Morphology, protein content, and inflammatory markers were assessed. NP cells isolated from healthy autopsy (Mongrel hounds) tissue were co-cultured with canine MCs within agarose constructs and treated with cromolyn sodium (CS) and PAR2 antagonist (PAR2A). Gene expression, sulfated glycosaminoglycan content, and stiffness of constructs were assessed. CD 31+ blood vessels, mast cell tryptase, and macrophage CD 163+ were increased in the degenerate surgical canine tissue compared to healthy autopsy. Pro-inflammatory genes were upregulated when canine NP cells were co-cultured with MCs and the stiffer microenvironment enhanced these effects. Treatment with CS and PAR2 inhibitors mediated key pro-inflammatory markers in canine NP cells. There is increased MC, MØs, and vascular ingrowth in the degenerate canine IVD tissue, similar to observations in the clinical population with IVDD and LBP. MCs co-cultured with canine NP cells drive inflammation, and CS and PAR2A are potential therapeutics that may mitigate the pathophysiology of IVDD in vitro.

Proteases in Irritable Bowel Syndrome: A Lot More Than Just Digestive Enzymes

The role of protease activation of inflammation in allergic respiratory diseases

The pathologic role of mast cell was classically studied in allergic diseases such as asthma and anaphylaxis.1 However, recently, the role of this multifunctional master cell has been actively investigated in the field of functional gastrointestinal disorder. Activation of mast cell has been suggested to increase intestinal permeability and promote visceral hypersensitivity in irritable bowel syndrome (IBS).2 As one of the possible mechanisms, release of tryptase from mast cell, activation of protease-activated receptor 2 (PAR-2), and subsequent release of neuropeptides from afferent neurons in the intestine have been proposed through several studies.3–5 In this issue of Gut and Liver, Liang et al.6 investigated the expression of tryptase, PAR-2 and several neuropeptides in the colonic mucosal of healthy control and patients with IBS. The authors found that mRNA expression of tryptase and PAR-2 was significantly elevated in the patients with IBS compared to the control. Tryptase protein expression was also elevated in the patients with IBS. However, PAR-2 protein expression did not differ between the IBS patients and the control. The protein level of calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P was significantly higher in the patients with diarrhea-type IBS (IBS-D) than in the control. This study added precious evidence which supports the role of mast cell in the pathogenesis of IBS. In addition, the elevation of all neuropeptides in IBS-D only well corresponds with the results of some studies which showed mast cell hyperplasia is more common in IBS-D.7,8 However, this study has some limitations. The interaction between mast cell and intestinal never is bidirectional.2 Neuropeptides can trigger activation of mast cell and conversely, activated mast cell can stimulate release of neuropeptides from nerve terminal. Therefore, elevation of both tryptase and neuropeptides in this study does not tell us which one is first. In addition, elevation of PAR-2 mRNA in the IBS patients did not led to the elevation of PAR-protein in this study. Another pathway which connects tryptase and the pathologic effects in IBS might be possible. Recently, the role of gut microbiota has been highlighted in the pathogenesis of IBS.9 Although there is still no convincing evidence, I think the change of gut microbiota might trigger activation of mast cell and subsequent visceral hypersensitivity. In-depth studies are required regarding the relationship between mast cell and the pathogenesis of IBS, considering the role of this tiny hero.

Proteinase activated receptor 2: a protean effector comes of age

Proteinase activated receptor 2 (PAR2) is a GPCR associated with inflammation, metabolism and disease. Clues to understanding how to block PAR2 signalling associated with disease without inhibiting PAR2 activation in normal physiology could be provided by studies of biased signalling. PAR2 ligand GB88 was profiled for PAR2 agonist and antagonist properties by several functional assays associated with intracellular G-protein-coupled signalling in vitro in three cell types and with PAR2-induced rat paw oedema in vivo. In HT29 cells, GB88 was a PAR2 antagonist in terms of Ca(2+) mobilization and PKC phosphorylation, but a PAR2 agonist in attenuating forskolin-induced cAMP accumulation, increasing ERK1/2 phosphorylation, RhoA activation, myosin phosphatase phosphorylation and actin filament rearrangement. In CHO-hPAR2 cells, GB88 inhibited Ca(2+) release, but activated G(i/o) and increased ERK1/2 phosphorylation. In human kidney tubule cells, GB88 inhibited cytokine secretion (IL6, IL8, GM-CSF, TNF-α) mediated by PAR2. A rat paw oedema induced by PAR2 agonists was also inhibited by orally administered GB88 and compared with effects of locally administered inhibitors of G-protein coupled pathways. GB88 is a biased antagonist of PAR2 that selectively inhibits PAR2/G(q/11)/Ca(2+)/PKC signalling, leading to anti-inflammatory activity in vivo, while being an agonist in activating three other PAR2-activated pathways (cAMP, ERK, Rho) in human cells. These findings highlight opportunities to design drugs to block specific PAR2-linked signalling pathways in disease, without blocking beneficial PAR2 signalling in normal physiology, and to dissect PAR2-associated mechanisms of disease in vivo.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor, mediating inflammation and pain signaling in neurons, thus it is considered to be a potential therapeutic target for inflammatory diseases. In this study, we performed a ligand-based virtual screening of 1.6 million compounds by employing a common-feature pharmacophore model and two-dimensional similarity search to identify a new PAR2 antagonist. The common-feature pharmacophore model was established based on the biological screening results of our in-house library. The initial virtual screening yielded a total number of 47 hits, and additional biological activity tests including PAR2 antagonism and anti-inflammatory effects resulted in a promising candidate, compound 43, which demonstrated an IC50 value of 8.22µM against PAR2. In next step, a PAR2 homology model was constructed using the crystal structure of the PAR1 as a template to explore the binding mode of the identified ligands. A molecular docking method was optimized by comparing the binding modes of a known PAR2 agonist GB110 and antagonist GB83, and applied to predict the binding mode of our hit compound 43. In-depth docking analyses revealed that the hydrophobic interaction with Phe243(5.39) is crucial for PAR2 ligands to exert antagonistic activity. MD simulation results supported the predicted docking poses that PAR2 antagonist blocked a conformational rearrangement of Na(+) allosteric site in contrast to PAR2 agonist that showed Na(+) relocation upon GPCR activation. In conclusion, we identified new a PAR2 antagonist together with its binding mode, which provides useful insights for the design and development of PAR2 ligands.

Proteases and protease-activated receptors (PARs) are major mediators involved in irritable bowel syndrome (IBS). Our objectives were to decipher the expression and functionality (calcium signaling) of PARs in human dorsal root ganglia (DRG) neurons and to define mechanisms involved in human sensory neuron signaling by IBS patient mediators. Human thoracic DRG were obtained from the national disease resource interchange. Expression of PAR1, PAR2, and PAR4 was assessed by immunohistochemistry and quantitative reverse transcription PCR (RT-qPCR) in whole DRG or in primary cultures of isolated neurons. Calcium signaling in response to PAR agonist peptides (PAR-AP), their inactive peptides (PAR-IP), thrombin (10 U/mL), supernatants from colonic biopsies of patients with IBS, or healthy controls, with or without PAR1 or PAR4 antagonist were studied in cultured human DRG neurons. PAR1, PAR2, and PAR4 were all expressed in human DRG, respectively, in 20%, 40%, and 40% of the sensory neurons. PAR1-AP increased intracellular calcium concentration in a dose-dependent manner. This increase was inhibited by PAR1 antagonism. By contrast, PAR2-AP, PAR4-AP, and PAR-IP did not cause calcium mobilization. PAR1-AP-induced calcium flux was significantly reduced by preincubation with PAR4-AP, but not with PAR2-AP. Thrombin increased calcium flux, which was inhibited by a PAR1 antagonist and increased by a PAR4 antagonist. Supernatants from colonic biopsies of patients with IBS induced calcium flux in human sensory neurons compared with healthy controls, and this induction was reversed by a PAR1 antagonist. Taken together, our results highlight that PAR1 antagonism should be investigated as a new therapeutic target for IBS symptoms.

We aimed to investigate the relationship between mast cell (MC) infiltration into the bladder with urothelial barrier dysfunction and bladder hyperactivity in a chronic bladder ischemia (CBI) rat model. We compared CBI rats (CBI group; n = 10) with normal rats (control group; n = 10). We measured the expression of mast cell tryptase (MCT) and protease-activated receptor 2 (PAR2), which are correlated with C fiber activation via MCT, and Uroplakins (UP Ia, Ib, II and III), which are critical to urothelial barrier function, via Western blotting. The effects of FSLLRY-NH2, a PAR2 antagonist, administered intravenously, on the bladder function of CBI rats were evaluated with a cystometrogram. In the CBI group, the MC number in the bladder was significantly greater (p = 0.03), and the expression of MCT (p = 0.02) and PAR2 (p = 0.02) was significantly increased compared to that of the control group. The 10 μg/kg FSLLRY-NH2 injection significantly increased the micturition interval of CBI rats (p = 0.03). The percentage of UP-II-positive cells on the urothelium with immunohistochemical staining was significantly lower in the CBI group than in the control group (p < 0.01). Chronic ischemia induces urothelial barrier dysfunction via impairing UP II, consequently inducing MC infiltration into the bladder wall and increased PAR2 expression. PAR2 activation by MCT may contribute to bladder hyperactivity.