Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-κB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor–2 (PAR₂). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR₂ antagonist and were absent in PAR₂-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR₂.

Mast cells (MCs) influence intercellular communication during inflammation by secreting cytoplasmic granules that contain diverse mediators. Here, we have demonstrated that MCs decode different activation stimuli into spatially and temporally distinct patterns of granule secretion. Certain signals, including substance P, the complement anaphylatoxins C3a and C5a, and endothelin 1, induced human MCs rapidly to secrete small and relatively spherical granule structures, a pattern consistent with the secretion of individual granules. Conversely, activating MCs with anti-IgE increased the time partition between signaling and secretion, which was associated with a period of sustained elevation of intracellular calcium and formation of larger and more heterogeneously shaped granule structures that underwent prolonged exteriorization. Pharmacological inhibition of IKK-β during IgE-dependent stimulation strongly reduced the time partition between signaling and secretion, inhibited SNAP23/STX4 complex formation, and switched the degranulation pattern into one that resembled degranulation induced by substance P. IgE-dependent and substance P–dependent activation in vivo also induced different patterns of mouse MC degranulation that were associated with distinct local and systemic pathophysiological responses. These findings show that cytoplasmic granule secretion from MCs that occurs in response to different activating stimuli can exhibit distinct dynamics and features that are associated with distinct patterns of MC-dependent inflammation.

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease‐activated receptor 2 (PAR₂) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR₂‐mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR₂, substance P (SP) and calcitonin gene‐related peptide (CGRP), mediators of pain transmission. In PAR₂‐expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR₂agonist enhanced Ca²⁺and current responses to the TRPV4 agonists phorbol ester 4α‐phorbol 12,13‐didecanoate (4αPDD) and hypotonic solutions. PAR₂‐agonist similarly sensitized TRPV4 Ca²⁺signals and currents in DRG neurons. Antagonists of phospholipase Cβ and protein kinases A, C and D inhibited PAR₂‐induced sensitization of TRPV4 Ca²⁺signals and currents. 4αPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR₂agonist sensitized TRPV4‐dependent peptide release. Intraplantar injection of PAR₂agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4αPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR₂agonist‐induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR₂activates a second messenger to sensitize TRPV4‐dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.