Pain 2016 Jul; 157(7):1541-50
Epac-protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation.
Gu Y, Li G, Chen Y, Huang L-Y M.
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX, 77555, USA
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 (PKCe) inside the cells. Protein kinase C epsilon has been established as the major PKC isoform mediating injury-induced hyperalgesic responses. On the other hand, the role of PKCa in receptor sensitization was seldom considered. Here, we studied the participation of PKCa 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 PKCa is also upregulated. Complete Freund adjuvant (CFA)–induced P2X3Rmediated hyperalgesia is not only blocked by Epac antagonists but also by the classical PKC isoform inhibitors, Go6976, and PKCa-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, PKCa is downstream of Epacs to mediate the enhancement of P2X3R responses in DRGs. The pattern of translocation of PKCa inside DRG neurons in response to CPT or CFA stimulation is distinct from that of PKCe. Thus, in contrast to prevalent view, PKCa also plays an essential role in producing complex inflammation-induced receptor-mediated hyperalgesia.
Rats with paw inflammation or peripheral nerve injuries show exaggerated nocifensive behaviors, e.g., fast paw withdrawals, repeated paw licking and rapid tail flicks, in response to mechanical pressure applied to the paw. This is a result of robust neuronal firing in response to a large amount of ATP released from damaged skin and nerve cells and dramatically enhanced ATP-induced P2X3R activity in injured DRGs (1,2). One of the causes for the enhanced P2X3R responses is a change in protein kinase activation induced by prostaglandin E2 (PGE2), an important modulator synthesized and released from tissues to modulate neuronal activity. Our studies show that under control conditions, PGE2 activates protein kinase A (PKA) to produce a moderate increase in P2X3R-mediated responses in DRGs (3). Following CFA-induced inflammation, PGE2 not only activates PKA, but also activates Epacs which evoke PKCe activation and produce a much enhanced P2X3R-mediated responses.
In addition to the evidence that PKCe enhances P2X3R responses after inflammation (3), PKCe has been found to sensitize thermal, mechanical, and chemical stimuli in DRGs (4). On the other hand, the participation of PKCa in generating hyperalgesia in DRGs has been largely ignored because an earlier study showed a lack of PKCa expression in cultured DRG cells harvested from neonatal rats (5). We and others in later studies found that PKCa is well expressed in DRGs isolated from various ages of rats (6-8). Here we present additional studies further characterizing PKCa expression in adult DRG neurons.
It has been well established that PKC translocates from cytoplasm to the cell membrane when it is activated (5). To determine the pattern of PKCa activation in cultured DRG neurons prepared from adult rats, we stained cells with anti-PKCa antibody and determined the distribution of PKCa in response to different activators. Following treatment of DRG neurons with the PKC activator, phorbol myristate acetate (PMA), all PKCa was permanently translocated to the cell membrane (Fig. 1). To determine if Epac can activate PKCa in DRG neurons, we applied CPT to cultured DRG neurons and determined PKCa distribution in those neurons. PKCa was found to be translocated from the cytoplasm to the cell membrane in a large percentage (~40%) of DRG neurons (Fig. 1). This observation suggests that Epac efficiently induces PKCa activation.
We then determined the co-localization of PKCa with P2X3Rs in DRG neurons by double immunostaining cells in DRG slices (Fig. 2). PKCa was found to be expressed in ~60% of small diameter (24±3 µm) DRG neurons. No large diameter cells were labeled. P2X3R labels were seen in ~27% of small diameter neurons. Thus, not all PKCa-containing neurons were stained positive for P2X3R. In contrast, all of the P2X3R positive stained neurons expressed PKCa. These observations suggest that PKCa modulates the activities of P2X3Rs, further supporting our conclusion that Epac-PKCa signaling is involved in inflammation-induced P2X3R-mediated hyperalgesia in DRGs.
Fig. 1. PKCa translocation in cultured DRG neurons. (A) Under a control (Con) condition, PKCa distributed evenly in the cytoplasm of DRG neurons. (B) PMA (1mM) induced membrane translocation of PKCa in all cells. (C) Activation of Epac by CPT (10mM) induced PKCa translocation from the cytoplasm to the cell membrane. Scale bar = 25 µm.
Fig. 2. pPKCa and P2X3R expression in DRG slices prepared from CFA-treated inflamed rats (A) pPKCa (green) and P2X3R (red) immunostaining in DRG neurons. Scale bar = 25 µm. (B) Cell number distribution of pPKCa and P2X3R expressed neurons. For pPKCa, data were obtained from analyses of 344 DRG neurons. For P2X3R, data were from 343 DRG neurons.
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