Investigating Mechanisms Regulating the In Vivo Actions of Delta Opioid Receptor Ligands

Abstract

Chronic pain and depression are widespread and debilitating diseases that, for many people, cannot be adequately addressed with current treatment options. Delta opioid receptor (DOR) agonists have been proposed as novel treatments for these disorders. DOR is a member of the opioid receptor family of G protein-coupled receptors (GPCRs). DOR signals through inhibitory Gαi/o proteins that are negatively regulated by regulator of G protein signaling (RGS) proteins. Activation of DOR induces antihyperalgesia and antidepressant-like effects in animal models without the constipation, respiratory depression, and abuse liability associated with mu opioid receptor agonists such as morphine. Unfortunately, some DOR agonists cause convulsions, hindering their development as therapeutics in humans. The experiments described in this thesis sought to further characterize the intracellular signaling pathways and mechanisms underlying DOR-mediated behaviors. Specifically, these studies used a number of mouse models to determine differences in the regulation of DOR-mediated convulsions relative to the antihyperalgesic and antidepressant-like effects of DOR agonists. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. Antidepressant-like effects were evaluated in the forced swim and tail suspension tests. Mice were also observed for convulsive activity post-agonist treatment. To assess the role of G protein signaling in DOR-mediated behaviors, we compared behaviors induced by the DOR agonist SNC80 in RGS4 knockout, Gαo RGS-insensitive (RGSi) knock-in, and Gαo knockout mice. SNC80-induced antihyperalgesia was enhanced in RGS4 knockout and Gαo RGSi mice. SNC80-induced antidepressant-like effects were also potentiated in both RGS4 knockout and Gαo RGSi mice. However, SNC80-induced convulsions were not changed in either strain. In Gαo heterozygous knockout mice, SNC80-induced antihyperalgesia was abolished while the antidepressant-like effects and convulsions were unaltered. Taken together, these data demonstrate that DOR-mediated antihyperalgesia and antidepressant-like effects, but not convulsions, are regulated by Gαo and RGS4. To further characterize the pharmacological properties mediating behavioral outcomes of DOR agonists, we compared the behavioral effects of SNC80 with those of the DOR partial agonist BU48. BU48 produced convulsions with similar potency to SNC80. BU48 also produced antidepressant-like effects with reduced potency relative to SNC80 and failed to elicit antihyperalgesia. These results suggest that the efficacy requirement for DOR-mediated convulsions may be low relative to other DOR-mediated behaviors. The efficacy requirements for DOR-mediated behaviors were further evaluated by comparing the shifts in the SNC80 dose response curves for each of these behaviors following decreases in DOR receptor reserve. Decreases in receptor reserve were produced using DOR heterozygous knockout mice as well as by treating mice with the DOR irreversible antagonist naltrindole-5’-isothiocyanate (5’-NTII). SNC80-induced antihyperalgesia displayed the largest potency shift in DOR heterozygous and 5’-NTII treated mice. Antidepressant-like effects displayed the next largest shift followed by convulsions. These findings suggest that DOR-mediated behaviors display the following rank order of efficacy requirement: convulsions < antidepressant like effects < antihyperalgesia. Furthermore, the DOR competitive antagonist naltrindole differentially shifted the SNC80 dose response curves of these behaviors, suggesting that different DOR receptor populations may mediate these behaviors. Overall, the work presented in this thesis suggests that DOR-mediated behaviors are generated by distinct signaling mechanisms and receptor populations. The possibility of pharmacologically targeting receptor populations or signaling pathways responsible for DOR-mediated analgesic and antidepressant-like effects without activating receptors that mediated convulsions should greatly aid the clinical viability of DOR agonists.