The importance of TRPV1-sensitisation factors for the development of neuropathic pain Natalia Malek, Agnieszka Pajak, Natalia Kolosowska, Mateusz Kucharczyk, Katarzyna Starowicz ⁎ Laboratory of Pain Pathophysiology, Department of Pain Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smetna 12 Street, 31-343 Krakow, Poland abstract article info Article history: Received 25 September 2014 Revised 20 January 2015 Accepted 2 February 2015 Available online 4 February 2015 Keywords: TRPV1 sensitisation DRG Neuropathic pain Neuronal inflammation CCI Transient receptor potential vanilloid type 1 (TRPV1), classically associated with transduction of high- temperature and low-pH pain, underlies pain hypersensitivity in neuropathic pain. The molecular regulation of TRPV1 channel activity is not yet fully understood. Therefore, we investigated factors regulating sensitisation of this receptor during development of neuropathic pain in a rat model of chronic construction injury (CCI) in the dorsal root ganglia (DRG). In the rat CCI model, elevated levels of pro-inflammatory cytokines (TNFα, IL-1β and IL-6) in DRG corresponded to development of neuropathic pain. We assessed the expression of known kinases influencing TRPV1 sensitisa- tion at the mRNA and/or protein level. Protein kinase C ε (PKCε) showed the strongest upregulation at the mRNA and protein levels among all tested kinases. Co-expression of PKCε and TRPV1 in L5 DRG of CCI animals was high during the development of neuropathic pain. The number of neurons expressing PKCε increased throughout the experiment. We provide complex data on the expression of a variety of factors involved in TRPV1 sensitisation in a CCI model of neuropathic pain. Our study supports evidence for involvement of TRPV1 in the development of neuropathic pain, by showing increased expression of interleukins and kinases responsible for the channel sensitisation. TNFα and NGF seem to play a role in the transition from acute to neuropathic pain, while PKCε in its maintenance. Further studies might confirm their significance as novel targets for the treatment of neuropathic pain. © 2015 Elsevier Inc. All rights reserved. 1. Background The increasing knowledge on the molecular biology of transient receptor potential vanilloid type 1 (TRPV1), a non-selective cation chan- nel, highlights its important role as a noxious signal integrator. It is expressed in both the peripheral and the central nervous system (CNS) and is involved in pain transduction, transmission, perception and modulation (Caterina et al., 2000). Dorsal root ganglia (DRG) contain TRPV1-expressing neurons (Hwang et al., 2005) that convey sensory information to the CNS. Activation of TRPV1, by external stimuli or endogenous agonists like anandamide (AEA), produces inward cation current, which may be involved in the sensitisation of nociceptive DRG neurons that causes hyperalgesia (Jara-Oseguera et al., 2008; Starowicz et al., 2008). Under pathological conditions, TRPV1 expression in noci- ceptive DRG neurons is altered in various models of peripheral neurop- athy (Biggs et al., 2007; Hudson et al., 2001; Kim et al., 2008; Rashid et al., 2003). The association of TRPV1 expression level with pain per- ception remains unclear, so the importance of channel sensitisation, manifested by the reduced activation threshold was postulated (reviewed in Palazzo et al., 2012). A number of modulators regulate TRPV1 activity and promote inflammatory or painful responses (Fig. 1). TRPV1 sensitisation is associ- ated with phosphorylation controlled by at least four serine/threonine ki- nases, including protein kinase A (PKA), protein kinase C (PKC), Ca 2+ / calmodulin-dependent kinase 2 (CaMK 2), and cyclin-dependent kinase 5 (Cdk5) (reviewed in Suh and Oh, 2005). Among these, PKA- and PKC- mediated phosphorylation has been widely investigated in various pain models (reviewed in Palazzo et al., 2012). Ahern and Premkumar (2002) reported that activation of PKC strongly sensitises the nociceptive response, which can be attenuated by both PKC inhibitors and mutations in TRPV1 phosphorylation sites. Particularly, PKCε modulation of TRPV1 activity plays a critical role in pain perception (Pan et al., 2010; Vellani et al., 2010; Ferrari et al., 2014). In contrast, the neuronal phosphatase cal- cineurin plays a major role in dephosphorylation of TRPV1 and is primar- ily responsible for the decrease in channel activity (Por et al., 2010). Moreover, as demonstrated by Ji et al. (2002), p38 mitogen-activated pro- tein kinase (P38-MAPK) activation, initiated by retrograde transport of nerve growth factor (NGF), acts to increase translocation of TRPV1 to the nociceptor membrane, thereby contributing to the maintenance of hypersensitivity. Indeed, the contribution of P38-MAPK to the progres- sion of neuropathic pain has been demonstrated in preclinical models Molecular and Cellular Neuroscience 65 (2015) 1–10 ⁎ Corresponding author. E-mail addresses: n.malek@if-pan.krakow.pl (N. Malek), pajak@if-pan.krakow.pl (A. Pajak), natkol@if-pan.krakow.pl (N. Kolosowska), mateusz.kucharczyk@uj-edu.pl (M. Kucharczyk), starow@if-pan.krakow.pl (K. Starowicz). http://dx.doi.org/10.1016/j.mcn.2015.02.001 1044-7431/© 2015 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne