Abstract
Analgesics targeting the δ-opioid receptor (DOR) may lead to fewer side effects than conventional opioid drugs, which mainly act on μ-opioid receptors (MOR), because of the less abundant expression of DOR in the CNS compared with MOR. Analgesic potential of DOR agonists increases after inflammation, an effect that may be mediated by DOR expressed in the peripheral sensory fibers. However, the expression of functional DOR at the plasma membrane of sensory neurons is controversial. Here we have used patch-clamp recordings and total internal reflection fluorescence microscopy to study the functional expression of DOR in sensory neurons from rat trigeminal (TG) and dorsal root ganglia (DRG). Real-time total internal reflection fluorescence microscopy revealed that treatment of TG and DRG cultures with the inflammatory mediator bradykinin (BK) caused robust trafficking of heterologously expressed GFP-tagged DOR to the plasma membrane. By contrast, treatment of neurons with the DOR agonist [d-Ala(2), d-Leu(5)]-enkephalin (DADLE) caused a decrease in the membrane abundance of DOR, suggesting internalization of the receptor after agonist binding. Patch-clamp experiments revealed that DADLE inhibited voltage-gated Ca(2+) channels (VGCCs) in 23% of small-diameter TG neurons. Pretreatment with BK resulted in more than twice as many DADLE responsive neurons (54%) but did not affect the efficacy of VGCC inhibition by DADLE. Our data suggest that inflammatory mediator-induced membrane insertion of DOR into the plasma membrane of peripheral sensory neurons may underlie increased DOR analgesia in inflamed tissue. Furthermore, the majority of BK-responsive TG neurons may have a potential to become responsive to DOR ligands in inflammatory conditions.
Highlights
Opioid drugs, such as morphine, are the current “gold standard” in pain relief, but their clinical applications are limited because of associated adverse side effects (Dickenson and Kieffer, 2006)
This, along with evidence for functional “priming” of DOR by the inflammatory mediator BK (Patwardhan et al, 2005), suggests that crosstalk between different proinflammatory GPCRs is important in opioid analgesia
Because Calcitonin gene-related peptide (CGRP) release is a Ca 2ϩ-dependent process that can be driven by the depolarization-induced activation of voltage-gated Ca 2ϩ channels (VGCCs) (Sakaguchi et al, 1991), it is plausible that increased inhibition of VGCC by Gi/o-coupled DOR may underlie the observed increase in efficacy of DOR-mediated antinociceptive action triggered by inflammation (Pradhan et al, 2013) or BK
Summary
Opioid drugs, such as morphine, are the current “gold standard” in pain relief, but their clinical applications are limited because of associated adverse side effects (Dickenson and Kieffer, 2006). In TIRF experiments, the effect of 10 nM DADLE on DOR-GFP fluorescence in DRG neurons was similar to that of 200 nM (Fig. 3B,C). Fura-2 Ca2ϩ imaging was performed here to determine what percentage of TG neurons responded to BK Transients similar to those observed in DRG were elicited in response to BK; 35 of 44 (79.5%) TG neurons responded to BK, suggesting that approximately three-fourths of the population of TG neurons (under culture conditions used here) express the BK receptor (Fig. 4A). In 26% of neurons (14 of 53), DADLE inhibited the Ca2ϩ transient significantly compared with control (the mean of 3 control transients, p Ͻ 0.05, paired t test, Fig. 5B,C) This experiment confirmed the pool size of TG neurons tonically expressing functional DOR. In 15 of 28 BK-pretreated TG neurons (30 min at 37°C), application of 200 nM DADLE inhibited the HVA Ca 2ϩ current in a rapid and ICa(pA)
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