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 (PAR(2)) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR(2)-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 PAR(2), substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR(2)-expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR(2) agonist enhanced Ca-2 and current responses to the TRPV4 agonists phorbol ester 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) and hypotonic solutions. PAR(2)-agonist similarly sensitized TRPV4 Ca-2 signals and currents in DRG neurons. Antagonists of phospholipase C beta and protein kinases A, C and D inhibited PAR(2)-induced sensitization of TRPV4 Ca-2 signals and currents. 4 alpha PDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR(2) agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR(2) agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4 alpha PDD and hypotonic solutions. Deletion of TRPV4 prevented PAR(2) agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR(2) 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.