Peripheral signaling mechanisms underlying mu‐delta opioid synergy

Abstract

In response to the ongoing opioid epidemic, and in an effort to develop new analgesics with improved safety profiles, researchers have invested significant energy into further understanding the various downstream signaling cascades initiated by opioid receptor activation. Recent studies have demonstrated that G protein-biased ligands demonstrate reduced side effects such as respiratory depression and tolerance. Further research has shown that bivalent or bifunctional opioid compounds can also result in differential signal transduction. We hypothesized that a combination of mu and delta opioid agonists would engage different signaling pathways than drugs that target only one opioid receptor. Therefore, we probed the signaling mechanisms underlying the behavioral analgesic synergy previously demonstrated by a 1:1 combination of loperamide, a peripherally-restricted mu-opioid agonist, and oxymorphindole, a delta-opioid agonist. Using the complete Freund's adjuvant model of inflammatory pain and the Hargreaves assay for thermal nociception, the anti-hyperalgesic effect of loperamide-oxymorphindole in male and female mice was challenged by co-administration of a recently developed antagonist against MOR-DOR heteromers, D24M. Then, mice were given an injection of loperamide, oxymorphindole, or their combination with and without pertussis toxin in order to assess the involvement of Gai/o signaling on the behavioral anti-hyperalgesia. Finally, in naïve mice, the role of G protein-coupled inwardly-rectifying potassium (GIRK) channels, a downstream target of G proteins, was investigated using the peptide inhibitor tertiapin-Q, as well as GIRK KO mice. The data demonstrate that D24M was significantly more potent in antagonizing the loperamide-oxymorphindole combination (p<0.01) than either drug alone, and that pertussis toxin significantly reduces the anti-hyperalgesic effect of the combination (p<0.05). Finally, both pharmacologic and genomic inhibition of GIRK2 function results in a significant reduction of the combination's anti-nociceptive effect (p<0.0001). From these data we conclude that the loperamide-oxymorphindole combination binds to heterodimeric opioid receptors, activates a G protein signaling pathway, and opens GIRK channels.