The tyrosine kinase inhibitor bafetinib inhibits PAR 2‐induced activation of TRPV 4 channels in vitro and pain in vivo

Background and Purpose Protease‐activated receptor 2 ( PAR 2) is expressed on nociceptive neurons, and can sensitize transient receptor potential ( TRP ) ion channels to amplify neurogenic inflammation and pain. The mechanisms by which this occurs are not fully understood. PAR 2 causes receptor‐operated activation of TRPV 4 channels and TRPV 4 null mice have attenuated PAR 2‐stimulated neurogenic inflammation and mechanical hyperalgesia. Here we investigate the intracellular signalling mechanisms underlying PAR 2‐induced TRPV 4 channel activation and pain. Experimental Approach Responses of non‐transfected and TRPV 4‐transfected HEK 293 cells to agonists of PAR2 (trypsin and SLIGRL ) and TRPV 4 channels ( GSK 1016790 A ) were determined using calcium imaging. Inhibitors of TRPV 4 channels ( HC 067047), sarcoendoplasmic reticulum calcium transport ATP ase (thapsigargin), G α q ( UBO‐QIC ), tyrosine kinases (bafetinib and dasatinib) or PI 3 kinases (wortmannin and LY 294002) were used to investigate signalling mechanisms. In vivo effects of tyrosine kinase inhibitors on PAR 2 ‐induced mechanical hyperalgesia were assessed in mice. Key Results I n non‐transfected HEK 293 cells, PAR2 activation transiently increased intracellular calcium ([ Ca 2+ ] i ). Functional expression of TRPV 4 channels caused a sustained increase of [ Ca 2+ ] i , inhibited by HC 067047, bafetinib and wortmannin; but not by thapsigargin, UBO‐QIC , dasatinib or LY 294002. B afetinib but not dasatinib inhibited PAR2 ‐induced mechanical hyperalgesia in vivo . Conclusions and Implications This study supports a role for tyrosine kinases in PAR2 ‐mediated receptor‐operated gating of TRPV 4 channels, independent of G α q stimulation. The ability of a tyrosine kinase inhibitor to diminish PAR2 ‐induced activation of TRPV 4 channels and consequent mechanical hyperalgesia identifies bafetinib (which is in development in oncology) as a potential novel analgesic therapy.