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. 2018 May;175(10):1760-1769.
doi: 10.1111/bph.14191. Epub 2018 Apr 6.

Exchange factor directly activated by cAMP-PKCε signalling mediates chronic morphine-induced expression of purine P2X3 receptor in rat dorsal root ganglia

Wenying Wang  1 , Xiaqing Ma  1   2 , Limin Luo  1 , Min Huang  1 , Jing Dong  1 , Xiaoli Zhang  1 , Wei Jiang  1 , Tao Xu  1   3
Affiliations

Exchange factor directly activated by cAMP-PKCε signalling mediates chronic morphine-induced expression of purine P2X3 receptor in rat dorsal root ganglia

Wenying Wang et al. Br J Pharmacol. 2018 May.

Abstract

Background and purpose: The P2X3 receptor is a major receptor in the processing of nociceptive information in dorsal root ganglia. We investigated the role of the P2X3 receptor and the detailed mechanisms underlying chronic morphine-induced analgesic tolerance in rats.

Experimental approach: Repeated i.t. morphine treatment was used to induce anti-nociceptive tolerance. The expression of spinal P2X3 receptor, phosphorylated PKCε and exchange factor directly activated by cAMP (Epac) were evaluated. Effects of A-317491 (P2X3 antagonist), ε-V1-2 (PKCε inhibitor) and ESI-09 (Epac inhibitor) on mechanical pain thresholds and tail-flick latency after chronic morphine treatment were determined. Co-localization of P2X3 receptor with NeuNs (marker of neuron), IB4 (marker of small DRG neurons), peripherin, PKCε and Epac were performed by double immunofluorescence staining.

Key results: Chronic morphine time-dependently increased the expression of P2X3 receptor, phosphorylated PKCε and Epac in DRGs. ε-V1-2 prevented chronic morphine-induced expression of P2X3 receptor. ESI-09 decreased the phosphorylation of PKCε and up-regulated expression of Epac after chronic morphine exposure. Mechanical pain thresholds and tail-flick latency showed that A317491, ε-V1-2 and ESI-09 significantly attenuated the loss of morphine's analgesic potency. Morphine-induced P2X3 receptor expression mainly occurred in neurons staining for IB4 and peripherin. Co-localization of P2X3 receptor with PKCε and Epac was demonstrated in the same neurons.

Conclusions and implications: Chronic morphine exposure increased the expression of P2X3 receptor, and i.t. P2X3 receptor antagonists attenuated the loss of morphine's analgesic effect. Inhibiting Epac/PKCε signalling was shown to play a significant inhibitory role in chronic morphine-induced P2X3 receptor expression and attenuate morphine-induced tolerance.

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Figures

Figure 1
Figure 1
(A) Expression of P2X3 receptor and (B) number of P2X3 receptor‐positive cells per section per animal in DRGs increase after repeated morphine treatment. The DRGs were removed 2 h after morphine injection on day 7 (n = 6,*P < 0.05 vs. saline (S) group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.
Figure 2
Figure 2
Daily i.t. injection of the selective P2X3 receptor antagonist A317491 attenuates morphine‐induced anti‐nociceptive tolerance. (A) Schematic of the experimental timeline. (B) Thermal and (C) mechanical thresholds measured daily in all groups (n = 6; *P < 0.05 vs. saline group; # P < 0.05 vs. morphine group).
Figure 3
Figure 3
Morphine‐induced P2X3 receptor up‐regulation is co‐localized with IB4 and peripherin in the DRG. (A) Confocal images of P2X3 receptor (green) and ionized Ca2+‐binding adapter molecule 1 (Iba1) (red) immunofluorescence with no co‐localization. (B) Immunofluorescence images of P2X3 receptor (red) and IB4 (green) co‐localization in DRGs. (C) There was less co‐localization of P2X3 receptor (green) with NF200 (red) in myelinated neurons. (D) Co‐localization of P2X3 receptor (green) with peripherin (red) in unmyelinated/small‐calibre neurons. DRG samples from the 7 day morphine group (scale bars: A/B, 80 μm; C/D, 20 μm).
Figure 4
Figure 4
Involvement of PKCε in morphine (Mor)‐induced antinociceptive tolerance and P2X3 receptor expression. (A) The phosphorylation of PKCε in the DRG increased time dependently with chronic Mor treatment. (B) The inhibitor of PKCε, ε‐V1‐2, reversed the increased P2X3 receptor expression induced by repeated Mor treatment. (C) PKCε co‐localized with P2X3 receptor in DRGs (scale bar: 50 μm for the up; 20 μm for the down). (D) Mor tolerance was attenuated when Mor was administered with ε‐V1‐2 for 7 days (n = 6,*P < 0.05 vs. saline (S) group; # P < 0.05 vs. morphine group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.
Figure 5
Figure 5
Epac is responsible for the chronic morphine (Mor)‐induced phosphorylation of PKCε and increase in P2X3 receptor expression. (A) The expression of Epac in the DRG increased time dependently after chronic Mor exposure. (B) The inhibitor of Epac, ESI‐09, reversed the increased P2X3 receptor expression and phosphorylation of PKCε induced by repeated morphine treatment. (C) Epac co‐localized with P2X3 receptor in DRGs (scale bar: 50 μm for the left; 20 μm for the right). (D) Morphine tolerance was attenuated by co‐administration of morphine and ESI‐09 for 7 days (n = 6,*P < 0.05 vs. saline (S) group; # P < 0.05 vs. morphine group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.
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