Mechanisms for enhancement of μ-opioid receptor-mediated antinociception in the central nervous system through gallein-dependent modulation of downstream G protein βγ subunit signaling.

The clinical utility of μ-opioid receptor (MOR) agonists is limited by side effects and tolerance. G protein βγ subunits (Gβγ) are critical mediators of MOR signaling that not only drive anticonception but also regulate phospholipase C and G protein-coupled receptor kinase pathways that feedback and inhibit MOR signaling. Modulation of Gβγ signaling with small molecules to preferentially inhibit feedback inhibition of MOR has been proposed as a strategy to enhance analgesia without exacerbating adverse effects. In this study, we investigated mechanisms by which gallein, a small-molecule Gβγ signaling modulator, enhances MOR-mediated antinociception in vivo. Gallein pretreatment significantly enhanced the antinociceptive effects of different MOR agonists and nonopioid analgesics that act through Gi-coupled G protein-coupled receptors, with the strongest effects observed upon coadministration with morphine. Gallein-mediated enhancement of morphine-induced antinociception was abolished by the opioid antagonist naloxone and was absent in MOR knockout mice. Notably, systemic naloxone methiodide, which cannot cross the blood-brain barrier, did not prevent gallein-mediated enhancement of morphine-induced antinociception. Intracerebroventricular, but not intrathecal, administration of gallein enhanced morphine-induced antinociception. Gallein was also effective at enhancing the action of morphine in a hot plate assay, implicating supraspinal MOR dependency. Lastly, 10 S/T-A mice expressing phosphosite-deficient MORs lost responsiveness to gallein treatment, supporting a mechanism of action in which gallein inhibits Gβγ-dependent inhibitory feedback phosphorylation of MOR in the central nervous system. SIGNIFICANCE STATEMENT: These findings provide insight into the mechanisms for potentiation of the antinociceptive effects of μ-opioid receptor (MOR) agonists via modulation of G protein βγ (Gβγ) signaling pathways. The study demonstrates dependence on central MORs and requires the ability of MOR to be feedback-regulated through phosphorylation. Pharmacological modulation of Gβγ signaling could serve as effective adjuncts to traditional opioid therapy.