Abstract
BACKGROUND: Proteases and histamine, co-secreted by mast cells and bacteria, sensitize colonic nociceptors and contribute to irritable bowel syndrome (IBS) pain. OBJECTIVE: To determine whether irreversible proteolytic cleavage of protease-activated receptor-2 (PAR (2) ) and its continued activity in endosomes amplify and sustain otherwise transient pronociceptive actions of histamine receptors (HRs) to cause recurrent pain, the defining symptom of IBS. DESIGN: We investigated the coexpression of PAR (2) and H (1) R in nociceptors using RNAscope in situ hybridization and assessed the consequences of coactivation using electrophysiological assays of nociceptor sensitization and biophysical measurements of receptor and effector activity. RESULTS: PAR (2) and H (1) R were coexpressed by human and mouse dorsal root ganglion nociceptors. Intracolonic infusion of fecal supernatants from IBS patients enhanced mechanosensitivity of colonic nociceptors in mice. Antagonists of PAR (2) or H (1-4) R abolished this response. Combined administration of subthreshold concentrations of trypsin and histamine replicated the effects of fecal supernatant and caused hyperexcitability of isolated nociceptors. Pre-activation of PAR (2) sensitized histamine-induced hyperexcitability. Endocytosis inhibitors prevented this hypersensitivity, consistent with sustained endosomal signaling of PAR (2) and persistent nociceptor hyperexcitability. Trypsin amplified histamine-induced activation of H (1) R and β-arrestin2 and Gαq effectors at the plasmalemma and in endosomes. Conversely, histamine did not sensitize trypsin-induced hyperexcitability of neurons, in line with the inability of histamine to induce sustained nociceptor hypersensitivity. CONCLUSIONS: By amplifying and maintaining the otherwise transient actions of H (1) R and possibly other pain receptors, persistent PAR (2) endosomal signaling makes a dominant contribution to IBS-related colonic pain. SUMMARY BOX: What is already known on this topic: Proteases and histamine are increased in IBS patients and cause visceral pain.What this study adds: Prolonged intracellular PAR (2) signaling sensitizes and maintains H (1) R activity to amplify and maintain pain. How this might affect research, practice or policy: Although neuroactive factors can act synergistically to amplify and maintain IBS pain, antagonists of dominant receptors ( e . g ., PAR (2) ) can provide effective treatment.