Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice

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 PAR₂ agonist 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 PAR₂ agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR₂ agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4αPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR₂ agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR₂ activates 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.