Grace M S, Lieu T, Darby B, Abogadie F C, Veldhuis N, Bunnett N W, McIntyre P
School of Medical Sciences and Health Innovations Research Institute, RMIT University, Bundoora, VIC, Australia.
Br J Pharmacol. 2014 Aug;171(16):3881-94. doi: 10.1111/bph.12750.
Protease-activated receptor 2 (PAR2) 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. PAR2 causes receptor-operated activation of TRPV4 channels and TRPV4 null mice have attenuated PAR2-stimulated neurogenic inflammation and mechanical hyperalgesia. Here we investigate the intracellular signalling mechanisms underlying PAR2-induced TRPV4 channel activation and pain.
Responses of non-transfected and TRPV4-transfected HEK293 cells to agonists of PAR2 (trypsin and SLIGRL) and TRPV4 channels (GSK1016790A) were determined using calcium imaging. Inhibitors of TRPV4 channels (HC067047), sarcoendoplasmic reticulum calcium transport ATPase (thapsigargin), Gαq (UBO-QIC), tyrosine kinases (bafetinib and dasatinib) or PI3 kinases (wortmannin and LY294002) were used to investigate signalling mechanisms. In vivo effects of tyrosine kinase inhibitors on PAR2 -induced mechanical hyperalgesia were assessed in mice.
In non-transfected HEK293 cells, PAR2 activation transiently increased intracellular calcium ([Ca(2+) ]i ). Functional expression of TRPV4 channels caused a sustained increase of [Ca(2+) ]i , inhibited by HC067047, bafetinib and wortmannin; but not by thapsigargin, UBO-QIC, dasatinib or LY294002. Bafetinib but not dasatinib inhibited PAR2-induced mechanical hyperalgesia in vivo.
This study supports a role for tyrosine kinases in PAR2-mediated receptor-operated gating of TRPV4 channels, independent of Gαq stimulation. The ability of a tyrosine kinase inhibitor to diminish PAR2-induced activation of TRPV4 channels and consequent mechanical hyperalgesia identifies bafetinib (which is in development in oncology) as a potential novel analgesic therapy.
蛋白酶激活受体2（PAR2）在伤害性神经元上表达，可使瞬时受体电位（TRP）离子通道敏感化，以放大神经源性炎症和疼痛。其发生机制尚未完全明确。PAR2可引起TRPV4通道的受体介导性激活，而TRPV4基因敲除小鼠的PAR2刺激的神经源性炎症和机械性痛觉过敏有所减轻。在此，我们研究PAR2诱导的TRPV4通道激活及疼痛背后的细胞内信号传导机制。
使用钙成像技术测定未转染及转染TRPV4的HEK293细胞对PAR2激动剂（胰蛋白酶和SLIGRL）及TRPV4通道激动剂（GSK1016790A）的反应。使用TRPV4通道抑制剂（HC067047）、肌浆网钙转运ATP酶抑制剂（毒胡萝卜素）、Gαq抑制剂（UBO-QIC）、酪氨酸激酶抑制剂（巴非替尼和达沙替尼）或PI3激酶抑制剂（渥曼青霉素和LY294002）来研究信号传导机制。评估酪氨酸激酶抑制剂对小鼠体内PAR2诱导的机械性痛觉过敏的影响。
在未转染的HEK293细胞中，PAR2激活可使细胞内钙（[Ca(2+)]i）短暂升高。TRPV4通道的功能性表达导致[Ca(2+)]i持续升高，HC067047、巴非替尼和渥曼青霉素可抑制此升高；但毒胡萝卜素、UBO-QIC、达沙替尼或LY294002则不能。巴非替尼而非达沙替尼可在体内抑制PAR2诱导的机械性痛觉过敏。
本研究支持酪氨酸激酶在PAR2介导的TRPV4通道受体介导性门控中发挥作用，且独立于Gαq刺激。酪氨酸激酶抑制剂能够减少PAR2诱导的TRPV4通道激活及随之而来的机械性痛觉过敏，这表明巴非替尼（正在肿瘤学领域进行研发）是一种潜在的新型镇痛疗法。
