Opioid agonist and antagonist treatment differentially regulates immunoreactive μ-opioid receptors and dynamin-2 in vivo

Abstract

Opioid agonists and antagonists can regulate the density of μ-opioid receptors in whole animal and in cell culture. High intrinsic efficacy agonists (e.g., etorphine), but not lower intrinsic efficacy agonists (e.g., morphine), produce μ-opioid receptor down-regulation and can alter the abundance of μ-opioid receptor mRNA. Conversely, opioid antagonists substantially increase the density of μ-opioid receptors without changing its mRNA. μ-Opioid receptor up-regulation has been associated with decreases in the trafficking protein dynamin-2, whereas μ-opioid receptor down-regulation produces an increase in dynamin-2 abundance. To probe the differences between opioid agonist and antagonist-induced μ-opioid receptor regulation, the current study determined changes in μ-opioid receptor density using a combined radioligand binding ([ 3H] DAMGO) and quantitative Western blotting approach in mouse spinal cord. Furthermore, the differences between intermittent and continuous dosing protocols were evaluated. Continuous (7–8 days) s.c. infusions of naloxone (5 mg/kg/day) or naltrexone (15 mg s.c. implant pellet) increased μ-opioid receptor density in radioligand binding assays (≈+80%) in mouse spinal cord and down-regulated dynamin-2 abundance (≈−30%), but had no effect on the abundance of immunoreactive μ-opioid receptor. Continuous (7 days) s.c. infusion of etorphine (200 μg/kg/day) decreased immunoreactive μ-opioid receptor (≈−35%) and [ 3H] DAMGO binding (≈−30%), and concurrently increased dynamin-2 abundance (≈+40%). Continuous (7 days) morphine infusion (40 mg/kg/day plus 25 mg s.c. implant pellet) had no effect on any outcome measure. Delivery of the same daily dose of etorphine or naloxone using intermittent (every 24 h for 7 days) s.c. administration had no effect on immunoreactive μ-opioid receptor, [ 3H] DAMGO binding or dynamin-2 abundance. These data indicate that μ-opioid receptor density, determined in radioligand binding assays, and immunoreactive dynamin-2 abundance are regulated by continuous, but not intermittent, opioid ligand treatment. Furthermore, the differential regulation of μ-opioid receptor abundance by agonists and antagonists in immunoblotting assays contrasts with changes in [ 3H] DAMGO binding. Taken together, these results suggest that etorphine-induced down-regulation may depend upon μ-opioid receptor degradation and changes in dynamin-2-mediated receptor trafficking. Conversely, antagonist-induced up-regulation does not require an increase in μ-opioid receptor synthesis and may entail conversion of receptors to an appropriate conformation to bind ligand, as well as changes in receptor trafficking.