These data demonstrate that inflammatory pain induced by CFA results in increased functionality of δORs in DRG neurons. This is shown by an increased inhibition of Ca2+ channels by the δOR agonist, SNC80, which mirrored an enhanced efficacy of SNC80 to inhibit mechanical allodynia. These data indicate that, regardless of the trafficking events that may or may not be involved, chronic inflammatory pain produces an enhanced responsivity of δORs.
In this study, we used δOR-VDCC coupling in DRGs as an ex vivo measure of δOR function that correlates with the pain-relieving effects of δOR agonists. We found that in a naïve, injury-free state the δOR agonist SNC80 did not alter the response threshold to von Frey filaments. However, following induction of inflammatory pain SNC80 potently inhibited CFA-induced allodynia. This in vivo gain of function was mirrored by an increased efficacy of SNC80 to inhibit Ca2+ channels within the DRGs. These results reflect previous work which has shown that compared to μ agonists, δ agonists are poor analgesics in acute pain  yet, they are highly effective in chronic inflammatory and neuropathic pain, likely due to an induction of δ receptor function following chronic pain [6, 28–31]. Further, the pain-relieving effects of δOR agonists has been previously shown to be mediated at the level of primary afferent neurons , supporting the notion that changes in Ca2+ channel coupling within the primary afferents would reflect behavioral responding. In addition, we had also shown a similar correlation following chronic use of δOR agonists, where uncoupling of δORs from VDCCs were observed following analgesic tolerance . However, as δOR-VDCC coupling in DRGs is one of several pathways activated by δOR agonists [32–34], it is likely that the analgesic effects of δOR agonists reflect the co-operative influence of these different signaling cascades that may include δOR inhibition of Ca2+ channels.
δORs are a member of the G
-coupled family of G-protein coupled receptors (GPCRs) and, although able to inhibit VDCCs in DRG neurons, δOR agonists have not been shown to produce significant VDCC inhibition in the basal state (Figure one, ). Chronic inflammatory pain increased δOR-VDCC inhibition which could have been a result of several factors. Likely candidates include; an increase in the number of receptor-complexes available for ligand activation; changes in the number or kinetics of Ca2+ channel recruitment by these activated receptors; or an altered signaling pathway by which δORs inhibit VDCCs. CFA has been shown previously to reduce Ca2+ channel density in small to medium sized (<40 μM) DRG neurons . However, we did not observe any effect of CFA on the voltage-dependent properties of Ca2+ currents in medium to large-sized DRG neurons. It is also unlikely that CFA induced an increase in receptor transcript and protein levels as neither have been reported to occur previously [18, 37]. However, as δORs have been found as signalosomes associated with their cognate G-proteins and other signaling molecules , it is possible that CFA altered the composition of these signalosomes. This may be in addition to, or independent of, an increase in the number of receptors on the cell membrane as previously suggested [6, 18, 39, 40]. Interestingly other paradigms such as treatment with bradykinin, chronic morphine, hypoxia and alcohol have also been shown to increase δOR function [33, 41–46] suggesting that δOR upregulation may have a number of clinically useful roles .
Internalized δORs are primarily targeted for degradation [7, 48–51] but some receptors may also be recycled  through the slow recycling, Rab11-dependent pathway . Several lines of evidence indicate that β-arrestin 2 mediates this trafficking of δORs following receptor internalization [25, 52] suggesting that we may have observed an altered response in β-arrestin 2 KOs. However, we found no effect of deleting β-arrestin 2 on the analgesic profile of SNC80, or on the enhanced δOR-VDCC coupling or VDCC density following CFA. However, we did observe that β-arrestin 2 plays a role in the contribution of N-type Ca2+ channels to the total Ca2+ current following CFA. Of the different types of VDCCs that contribute to the high voltage-activated currents in DRGs, the N-type normally contributes ~50% of the current . In KO neurons, this decreased to ~35% suggesting an increase in the contribution of R or P/Q type channels so as to maintain total current density. This raises an intriguing possibility that β-arrestins may regulate the contribution of Ca2+ channels to the total current following CFA.