A240 OPIOID-INDUCED HYPEREXCITABILITY IN DRG NEURONS IS MEDIATED BY DOR-DEPENDENT ENDOCYTOSIS

Abstract

Abstract Background Opioids are effective for the treatment of abdominal pain but are also associated with tolerance, and increased dosing leads to severe side effects. We previously showed that prolonged exposure to high doses of opioids evoked paradoxical hyperexcitability of colonic afferent nerves that was mediated by δ-opioid receptor (DOR) signaling. Recent studies suggest that DOR-dependent analgesia is mediated not only by G proteins but also via receptor endocytosis and downstream signaling, but it is unclear what intracellular signaling mechanisms are underlying opioid-induced hyperexcitability. Aims To examine the mechanisms underlying DOR-mediated hyperexcitability of dorsal root ganglia (DRG) neurons. Methods We assessed the excitability of DRG neurons isolated from C57BL/6 mice by measuring the rheobase (minimal current to elicit an action potential, i.e. lower rheobase=increased excitability) using perforated patch-clamp recordings. Dissociated neurons were exposed to a high concentration (10 µM) of the μ-opioid receptor agonist DAMGO, the DOR agonist DADLE, or the weakly internalizing DOR agonist ARM390 overnight. To examine the role of receptor endocytosis and intracellular receptor activation underlying the excitatory effect by opioids, DRG neurons exposed to DAMGO or DADLE were preincubated with the membrane-permeable opioid receptor antagonist naloxone or the endocytosis inhibitor Pitstop2. To further understand the mechanisms involved in the hyperexcitability evoked by opioid re-exposure, following overnight incubation with high concentrations of DAMGO or DADLE, neurons were washed for 1 hr and treated either with the PKA inhibitor H89 or the PKC inhibitor GFX before re-exposure to DAMGO or DADLE at a low concentration (10 nM). Results Neurons exposed to 10 µM DAMGO or DADLE were hyperexcitable (rheobase decreased 25 % and 26 % compared to controls respectively; p≤0.05, 2-way ANOVA). Naloxone and Pitstop2 blocked the increased excitability of DRG neurons induced by overnight incubations with either DAMGO or DADLE. In contrast to the hyperexcitability induced by DAMGO and DADLE, overnight incubation with 10 µM ARM390 decreased excitability (rheobase increased 31%, p≤0.05, unpaired t-test). The hyperexcitability induced by DAMGO and DADLE was reversed after a 1 hr washout but acute reapplication of a low concentration of DAMGO or DADLE (10nM) now evoked hyperexcitability (rheobase decreased 34 and 35 % respectively, p≤0.05, 2-way ANOVA). This effect was prevented by inhibiting PKC but not PKA. Conclusions Our data suggest that the DOR-dependent hyperexcitability evoked by prolonged exposure to high concentrations of opioids is dependent on receptor endocytosis and downstream PKC signaling. Targeting these pathways could mitigate the hyperexcitability of pain signaling neurons caused by high doses of opioids. Funding Agencies CCC