Epac-protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation

Sensitization of purinergic P2X3 receptors (P2X3Rs) is a major mechanism contributing to injury-induced exaggerated pain responses. We showed in a previous study that cyclic adenosine monophosphate (cAMP)-dependent guanine nucleotide exchange factor 1 (Epac1) in rat sensory dorsal root ganglia (DRGs) is upregulated after inflammatory injury, and it plays a critical role in P2X3R sensitization by activating protein kinase C epsilon (PKC epsilon) inside the cells. protein kinase Cepsilon has been established as the major PKC isoform mediating injury-induced hyperalgesic responses. On the other hand, the role of PKC alpha in receptor sensitization was seldom considered. Here, we studied the participation of PKC alpha in Epac signaling in P2X3R-mediated hyperalgesia. The expression of both Epac1 and Epac2 and the level of cAMP in DRGs are greatly enhanced after complete Freund adjuvant (CFA)-induced inflammation. The expression of phosphorylated PKC alpha is also upregulated. Complete Freund adjuvant (CFA)-induced P2X3R-mediated hyperalgesia is not only blocked by Epac antagonists but also by the classical PKC isoform inhibitors, Go6976, and PKC alpha-siRNA. These CFA effects are mimicked by the application of the Epac agonist, 8-(4-chlorophenylthio)-2 -O-methyl-cAMP (CPT), in control rats, further confirming the involvement of Epacs. Because the application of Go6976 prior to CPT still reduces CPT-induced hyperalgesia, PKC alpha is downstream of Epacs to mediate the enhancement of P2X3R responses in DRGs. The pattern of translocation of PKC alpha inside DRG neurons in response to CPT or CFA stimulation is distinct from that of PKC epsilon. Thus, in contrast to prevalent view, PKC alpha also plays an essential role in producing complex inflammation-induced receptor-mediated hyperalgesia.