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.

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