The mechanisms of inflammatory pain need to be identified in order to find new superior treatments. Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) are highly co-expressed in dorsal root ganglion neurons and implicated in pain development. Here, we examined the role of spinal PAR2 in hyperalgesia and the modulation of synaptic transmission in carrageenan-induced peripheral inflammation, using intrathecal (i.t.) treatment in the behavioral experiments and recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs and eEPSCs) in spinal cord slices. Intrathecal PAR2-activating peptide (AP) administration aggravated the carrageenan-induced thermal hyperalgesia, and this was prevented by a TRPV1 antagonist (SB 366791) and staurosporine i.t. pretreatment. Additionally, the frequency of the mEPSC and sEPSC and the amplitude of the eEPSC recorded from the superficial dorsal horn neurons were enhanced after acute PAR2 AP application, while prevented with SB 366791 or staurosporine pretreatment. PAR2 antagonist application reduced the thermal hyperalgesia and decreased the frequency of mEPSC and sEPSC and the amplitude of eEPSC. Our findings highlight the contribution of spinal PAR2 activation to carrageenan-induced hyperalgesia and the importance of dorsal horn PAR2 and TRPV1 receptor interactions in the modulation of nociceptive synaptic transmission.

Laboratory of Pain Research Institute of Physiology Czech Academy of Sciences Videnska 1083 142 20 Prague 4 Czech Republic

Zobrazit více v PubMed

Nystedt S., Emilsson K., Wahlestedt C., Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc. Natl. Acad. Sci. USA. 1994;91:9208–9212. doi: 10.1073/pnas.91.20.9208. PubMed DOI PMC

Nystedt S., Ramakrishnan V., Sundelin J. The proteinase-activated receptor 2 is induced by inflammatory mediators in human endothelial cells. Comparison with the thrombin receptor. J. Biol. Chem. 1996;271:14910–14915. doi: 10.1074/jbc.271.25.14910. PubMed DOI

Kelso E.B., Lockhart J.C., Hembrough T., Dunning L., Plevin R., Hollenberg M.D., Sommerhoff C.P., McLean J.S., Ferrell W.R. Therapeutic promise of proteinase-activated receptor-2 antagonism in joint inflammation. J. Pharmacol. Exp. Ther. 2006;316:1017–1024. doi: 10.1124/jpet.105.093807. PubMed DOI

Vergnolle N., Hollenberg M.D., Sharkey K.A., Wallace J.L. Characterization of the inflammatory response to proteinase-activated receptor-2 (PAR2)-activating peptides in the rat paw. Br. J. Pharmacol. 1999;127:1083–1090. doi: 10.1038/sj.bjp.0702634. PubMed DOI PMC

Steinhoff M., Vergnolle N., Young S.H., Tognetto M., Amadesi S., Ennes H.S., Trevisani M., Hollenberg M.D., Wallace J.L., Caughey G.H., et al. Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism. Nat. Med. 2000;6:151–158. doi: 10.1038/72247. PubMed DOI

Bunnett N.W. Protease-activated receptors: How proteases signal to cells to cause inflammation and pain. Semin Thromb Hemost. 2006;32(Suppl. 1):39–48. doi: 10.1055/s-2006-939553. PubMed DOI

Jimenez-Vargas N.N., Pattison L.A., Zhao P., Lieu T., Latorre R., Jensen D.D., Castro J., Aurelio L., Le G.T., Flynn B., et al. Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome. Proc. Natl. Acad. Sci. USA. 2018;115:E7438–E7447. doi: 10.1073/pnas.1721891115. PubMed DOI PMC

Ossovskaya V.S., Bunnett N.W. Protease-activated receptors: Contribution to physiology and disease. Physiol. Rev. 2004;84:579–621. doi: 10.1152/physrev.00028.2003. PubMed DOI

Zhao P., Lieu T., Barlow N., Metcalf M., Veldhuis N.A., Jensen D.D., Kocan M., Sostegni S., Haerteis S., Baraznenok V., et al. Cathepsin S causes inflammatory pain via biased agonism of PAR2 and TRPV4. J. Biol. Chem. 2014;289:27215–27234. doi: 10.1074/jbc.M114.599712. PubMed DOI PMC

Lieu T., Savage E., Zhao P., Edgington-Mitchell L., Barlow N., Bron R., Poole D.P., McLean P., Lohman R.J., Fairlie D.P., et al. Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor-2. Br. J. Pharmacol. 2016;173:2752–2765. doi: 10.1111/bph.13554. PubMed DOI PMC

Bohm S.K., Kong W., Bromme D., Smeekens S.P., Anderson D.C., Connolly A., Kahn M., Nelken N.A., Coughlin S.R., Payan D.G., et al. Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2. Biochem. J. 1996;314:1009–1016. doi: 10.1042/bj3141009. PubMed DOI PMC

Kanke T., Ishiwata H., Kabeya M., Saka M., Doi T., Hattori Y., Kawabata A., Plevin R. Binding of a highly potent protease-activated receptor-2 (PAR2) activating peptide, [3H]2-furoyl-LIGRL-NH2, to human PAR2. Br. J. Pharmacol. 2005;145:255–263. doi: 10.1038/sj.bjp.0706189. PubMed DOI PMC

Ricks T.K., Trejo J. Phosphorylation of protease-activated receptor-2 differentially regulates desensitization and internalization. J. Biol. Chem. 2009;284:34444–34457. doi: 10.1074/jbc.M109.048942. PubMed DOI PMC

Kumar P., Lau C.S., Mathur M., Wang P., DeFea K.A. Differential effects of beta-arrestins on the internalization, desensitization and ERK1/2 activation downstream of protease activated receptor-2. Am. J. Physiol. Cell Physiol. 2007;293:C346–C357. doi: 10.1152/ajpcell.00010.2007. PubMed DOI

Amadesi S., Nie J., Vergnolle N., Cottrell G.S., Grady E.F., Trevisani M., Manni C., Geppetti P., McRoberts J.A., Ennes H., et al. Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia. J. Neurosci. 2004;24:4300–4312. doi: 10.1523/JNEUROSCI.5679-03.2004. PubMed DOI PMC

Dai Y., Moriyama T., Higashi T., Togashi K., Kobayashi K., Yamanaka H., Tominaga M., Noguchi K. Proteinase-activated receptor 2-mediated potentiation of transient receptor potential vanilloid subfamily 1 activity reveals a mechanism for proteinase-induced inflammatory pain. J. Neurosci. 2004;24:4293–4299. doi: 10.1523/JNEUROSCI.0454-04.2004. PubMed DOI PMC

Salzer I., Ray S., Schicker K., Boehm S. Nociceptor Signalling through ion Channel Regulation via GPCRs. Int. J. Mol. Sci. 2019;20:2488. doi: 10.3390/ijms20102488. PubMed DOI PMC

DeFea K.A., Zalevsky J., Thoma M.S., Déry O., Mullins R.D., Bunnett N.W. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J. Cell Biol. 2000;148:1267–1281. doi: 10.1083/jcb.148.6.1267. PubMed DOI PMC

Stalheim L., Ding Y., Gullapalli A., Paing M.M., Wolfe B.L., Morris D.R., Trejo J. Multiple independent functions of arrestins in the regulation of protease-activated receptor-2 signaling and trafficking. Mol. Pharmacol. 2005;67:78–87. doi: 10.1124/mol.104.006072. PubMed DOI

Zhao P., Metcalf M., Bunnett N.W. Biased signaling of protease-activated receptors. Front. Endocrinol. 2014;5:67. doi: 10.3389/fendo.2014.00067. PubMed DOI PMC

Alier K.A., Endicott J.A., Stemkowski P.L., Cenac N., Cellars L., Chapman K., Andrade-Gordon P., Vergnolle N., Smith P.A. Intrathecal administration of proteinase-activated receptor-2 agonists produces hyperalgesia by exciting the cell bodies of primary sensory neurons. J. Pharmacol. Exp. Ther. 2008;324:224–233. doi: 10.1124/jpet.107.129171. PubMed DOI

Caterina M.J., Leffler A., Malmberg A.B., Martin W.J., Trafton J., Petersen-Zeitz K.R., Koltzenburg M., Basbaum A.I., Julius D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 2000;288:306–313. doi: 10.1126/science.288.5464.306. PubMed DOI

Spicarova D., Nerandzic V., Palecek J. Update on the role of spinal cord TRPV1 receptors in pain modulation. Physiol. Res. 2014;63:S225–S236. doi: 10.33549/physiolres.932713. PubMed DOI

Amadesi S., Cottrell G.S., Divino L., Chapman K., Grady E.F., Bautista F., Karanjia R., Barajas-Lopez C., Vanner S., Vergnolle N., et al. Protease-activated receptor 2 sensitizes TRPV1 by protein kinase Cepsilon- and A-dependent mechanisms in rats and mice. J. Physiol. 2006;575:555–571. doi: 10.1113/jphysiol.2006.111534. PubMed DOI PMC

Spicarova D., Palecek J. The role of the TRPV1 endogenous agonist N-Oleoyldopamine in modulation of nociceptive signaling at the spinal cord level. J. Neurophysiol. 2009;102:234–243. doi: 10.1152/jn.00024.2009. PubMed DOI

Spicarova D., Palecek J. Tumor necrosis factor alpha sensitizes spinal cord TRPV1 receptors to the endogenous agonist N-oleoyldopamine. J. Neuroinflamm. 2010;7:49. doi: 10.1186/1742-2094-7-49. PubMed DOI PMC

Spicarova D., Adamek P., Kalynovska N., Mrozkova P., Palecek J. TRPV1 receptor inhibition decreases CCL2-induced hyperalgesia. Neuropharmacology. 2014;81:75–84. doi: 10.1016/j.neuropharm.2014.01.041. PubMed DOI

Koetzner L., Gregory J.A., Yaksh T.L. Intrathecal protease-activated receptor stimulation produces thermal hyperalgesia through spinal cyclooxygenase activity. J. Pharmacol. Exp. Ther. 2004;311:356–363. doi: 10.1124/jpet.104.069484. PubMed DOI

Grant A.D., Cottrell G.S., Amadesi S., Trevisani M., Nicoletti P., Materazzi S., Altier C., Cenac N., Zamponi G.W., Bautista-Cruz F., et al. Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice. J. Physiol. 2007;578:715–733. doi: 10.1113/jphysiol.2006.121111. PubMed DOI PMC

Dai Y., Wang S., Tominaga M., Yamamoto S., Fukuoka T., Higashi T., Kobayashi K., Obata K., Yamanaka H., Noguchi K. Sensitization of TRPA1 by PAR2 contributes to the sensation of inflammatory pain. J. Clin. Investig. 2007;117:1979–1987. doi: 10.1172/JCI30951. PubMed DOI PMC

Chen Y., Yang C., Wang Z.J. Proteinase-activated receptor 2 sensitizes transient receptor potential vanilloid 1, transient receptor potential vanilloid 4, and transient receptor potential ankyrin 1 in paclitaxel-induced neuropathic pain. Neuroscience. 2011;193:440–451. doi: 10.1016/j.neuroscience.2011.06.085. PubMed DOI

Linley J.E., Rose K., Patil M., Robertson B., Akopian A.N., Gamper N. Inhibition of M current in sensory neurons by exogenous proteases: A signaling pathway mediating inflammatory nociception. J. Neurosci. 2008;28:11240–11249. doi: 10.1523/JNEUROSCI.2297-08.2008. PubMed DOI PMC

Jin X., Shah S., Liu Y., Zhang H., Lees M., Fu Z., Lippiat J.D., Beech D.J., Sivaprasadarao A., Baldwin S.A., et al. Activation of the Cl− channel ANO1 by localized calcium signals in nociceptive sensory neurons requires coupling with the IP3 receptor. Sci. Signal. 2013;6:ra73. doi: 10.1126/scisignal.2004184. PubMed DOI PMC

Mrozkova P., Spicarova D., Palecek J. Hypersensitivity Induced by Activation of Spinal Cord PAR2 Receptors Is Partially Mediated by TRPV1 Receptors. PLoS ONE. 2016;11:e0163991. doi: 10.1371/journal.pone.0163991. PubMed DOI PMC

Mrozkova P., Palecek J., Spicarova D. The role of protease-activated receptor type 2 in nociceptive signaling and pain. Physiol. Res. 2016;65:357–367. doi: 10.33549/physiolres.933269. PubMed DOI

Huang Z., Tao K., Zhu H., Miao X., Wang Z., Yu W., Lu Z. Acute PAR2 activation reduces GABAergic inhibition in the spinal dorsal horn. Brain Res. 2011;1425:20–26. doi: 10.1016/j.brainres.2011.09.058. PubMed DOI

Noorbakhsh F., Tsutsui S., Vergnolle N., Boven L.A., Shariat N., Vodjgani M., Warren K.G., Andrade-Gordon P., Hollenberg M.D., Power C. Proteinase-activated receptor 2 modulates neuroinflammation in experimental autoimmune encephalomyelitis and multiple sclerosis. J. Exp. Med. 2006;203:425–435. doi: 10.1084/jem.20052148. PubMed DOI PMC

Cenac N., Coelho A.M., Nguyen C., Compton S., Andrade-Gordon P., MacNaughton W.K., Wallace J.L., Hollenberg M.D., Bunnett N.W., Garcia-Villar R., et al. Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2. Am. J. Pathol. 2002;161:1903–1915. doi: 10.1016/S0002-9440(10)64466-5. PubMed DOI PMC

Radulovic M., Yoon H., Wu J., Mustafa K., Fehlings M.G., Scarisbrick I.A. Genetic targeting of protease activated receptor 2 reduces inflammatory astrogliosis and improves recovery of function after spinal cord injury. Neurobiol. Dis. 2015;83:75–89. doi: 10.1016/j.nbd.2015.08.021. PubMed DOI PMC

Ferrell W.R., Lockhart J.C., Kelso E.B., Dunning L., Plevin R., Meek S.E., Smith A.J., Hunter G.D., McLean J.S., McGarry F., et al. Essential role for proteinase-activated receptor-2 in arthritis. J. Clin. Investig. 2003;111:35–41. doi: 10.1172/JCI16913. PubMed DOI PMC

Vergnolle N., Bunnett N.W., Sharkey K.A., Brussee V., Compton S.J., Grady E.F., Cirino G., Gerard N., Basbaum A.I., Andrade-Gordon P., et al. Proteinase-activated receptor-2 and hyperalgesia: A novel pain pathway. Nat. Med. 2001;7:821–826. doi: 10.1038/89945. PubMed DOI

Cenac N. Protease-activated receptors as therapeutic targets in visceral pain. Curr. Neuropharmacol. 2013;11:598–605. doi: 10.2174/1570159X113119990039. PubMed DOI PMC

Velázquez K.T., Mohammad H., Sweitzer S.M. Protein kinase C in pain: Involvement of multiple isoforms. Pharmacol. Res. 2007;55:578–589. doi: 10.1016/j.phrs.2007.04.006. PubMed DOI PMC

Honore P., Wismer C.T., Mikusa J., Zhu C.Z., Zhong C., Gauvin D.M., Gomtsyan A., El Kouhen R., Lee C.H., Marsh K., et al. A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel transient receptor potential type V1 receptor antagonist, relieves pathophysiological pain associated with inflammation and tissue injury in rats. J. Pharmacol. Exp. Ther. 2005;314:410–421. doi: 10.1124/jpet.105.083915. PubMed DOI

Cui M., Honore P., Zhong C., Gauvin D., Mikusa J., Hernandez G., Chandran P., Gomtsyan A., Brown B., Bayburt E.K., et al. TRPV1 receptors in the CNS play a key role in broad-spectrum analgesia of TRPV1 antagonists. J. Neurosci. 2006;26:9385–9393. doi: 10.1523/JNEUROSCI.1246-06.2006. PubMed DOI PMC

Yu L., Yang F., Luo H., Liu F.Y., Han J.S., Xing G.G., Wan Y. The role of TRPV1 in different subtypes of dorsal root ganglion neurons in rat chronic inflammatory nociception induced by complete Freund’s adjuvant. Mol. Pain. 2008;4:61. doi: 10.1186/1744-8069-4-61. PubMed DOI PMC

Huang Y., Chen S.R., Chen H., Pan H.L. Endogenous transient receptor potential ankyrin 1 and vanilloid 1 activity potentiates glutamatergic input to spinal lamina I neurons in inflammatory pain. J. Neurochem. 2019;149:381–398. doi: 10.1111/jnc.14677. PubMed DOI PMC

Davis J.B., Gray J., Gunthorpe M.J., Hatcher J.P., Davey P.T., Overend P., Harries M.H., Latcham J., Clapham C., Atkinson K., et al. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature. 2000;405:183–187. doi: 10.1038/35012076. PubMed DOI

Wang Y., Gao Y., Tian Q., Deng Q., Zhou T., Liu Q., Mei K., Liu H., Ma R., Ding Y., et al. TRPV1 SUMOylation regulates nociceptive signaling in models of inflammatory pain. Nat. Commun. 2018;9:1529. doi: 10.1038/s41467-018-03974-7. PubMed DOI PMC

Gregus A.M., Doolen S., Dumlao D.S., Buczynski M.W., Takasusuki T., Fitzsimmons B.L., Hua X.Y., Taylor B.K., Dennis E.A., Yaksh T.L. Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors. Proc. Natl. Acad. Sci. USA. 2012;109:6721–6726. doi: 10.1073/pnas.1110460109. PubMed DOI PMC

Tohda C., Sasaki M., Konemura T., Sasamura T., Itoh M., Kuraishi Y. Axonal transport of VR1 capsaicin receptor mRNA in primary afferents and its participation in inflammation-induced increase in capsaicin sensitivity. J. Neurochem. 2001;76:1628–1635. doi: 10.1046/j.1471-4159.2001.00193.x. PubMed DOI

Abooj M., Bishnoi M., Bosgraaf C.A., Premkumar L.S. Changes in Spinal Cord Following Inflammatory and Neuropathic Pain and the Effectiveness of Resiniferatoxin. Open Pain J. 2016;9:1–14. doi: 10.2174/1876386301609010001. DOI

Ren K., Dubner R. Inflammatory Models of Pain and Hyperalgesia. ILAR J. 1999;40:111–118. doi: 10.1093/ilar.40.3.111. PubMed DOI

Soh U.J., Dores M.R., Chen B., Trejo J. Signal transduction by protease-activated receptors. Br. J. Pharmacol. 2010;160:191–203. doi: 10.1111/j.1476-5381.2010.00705.x. PubMed DOI PMC

Cesare P., Moriondo A., Vellani V., McNaughton P.A. Ion channels gated by heat. Proc. Natl. Acad. Sci. USA. 1999;96:7658–7663. doi: 10.1073/pnas.96.14.7658. PubMed DOI PMC

Ding-Pfennigdorff D., Averbeck B., Michaelis M. Stimulation of PAR-2 excites and sensitizes rat cutaneous C-nociceptors to heat. Neuroreport. 2004;15:2071–2075. doi: 10.1097/00001756-200409150-00015. PubMed DOI

Bao Y., Hou W., Yang L., Liu R., Gao Y., Kong X., Shi Z., Li W., Zheng H., Jiang S., et al. Increased expression of protease-activated receptor 2 and 4 within dorsal root ganglia in a rat model of bone cancer pain. J. Mol. Neurosci. 2015;55:706–714. doi: 10.1007/s12031-014-0409-1. PubMed DOI

Bao Y., Hou W., Liu R., Gao Y., Kong X., Yang L., Shi Z., Li W., Zheng H., Jiang S., et al. PAR2-mediated upregulation of BDNF contributes to central sensitization in bone cancer pain. Mol. Pain. 2014;10:28. doi: 10.1186/1744-8069-10-28. PubMed DOI PMC

Chen K., Zhang Z.F., Liao M.F., Yao W.L., Wang J., Wang X.R. Blocking PAR2 attenuates oxaliplatin-induced neuropathic pain via TRPV1 and releases of substance P and CGRP in superficial dorsal horn of spinal cord. J. Neurol. Sci. 2015;352:62–67. doi: 10.1016/j.jns.2015.03.029. PubMed DOI

Chen D., Liu N., Li M., Liang S. Blocking PAR2 Alleviates Bladder Pain and Hyperactivity via TRPA1 Signal. Transl. Neurosci. 2016;7:133–138. doi: 10.1515/tnsci-2016-0020. PubMed DOI PMC

Lucena F., McDougall J.J. Pain responses to protease-activated receptor-2 stimulation in the spinal cord of naïve and arthritic rats. Neurosci. Lett. 2020;739:135391. doi: 10.1016/j.neulet.2020.135391. PubMed DOI

Spicarova D., Nerandzic V., Palecek J. Modulation of spinal cord synaptic activity by tumor necrosis factor α in a model of peripheral neuropathy. J. Neuroinflamm. 2011;8:177. doi: 10.1186/1742-2094-8-177. PubMed DOI PMC

Uchytilova E., Spicarova D., Palecek J. TRPV1 antagonist attenuates postoperative hypersensitivity by central and peripheral mechanisms. Mol. Pain. 2014;10:67. doi: 10.1186/1744-8069-10-67. PubMed DOI PMC

Inhibition of synaptic transmission by anandamide precursor 20:4-NAPE is mediated by TRPV1 receptors under inflammatory conditions

Hypersensitivity Induced by Intrathecal Bradykinin Administration Is Enhanced by N-oleoyldopamine (OLDA) and Prevented by TRPV1 Antagonist