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

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.