Inhibition of Agonist-Induced Down-Regulation of the δ-Opioid Receptor with a Proteasome Inhibitor Attenuates Opioid Tolerance in Human Embryonic Kidney 293 Cells

This study was designed to test the hypothesis that inhibition of agonist-induced δ-receptor down-regulation would block the development of opioid tolerance in a cell-based model. A human embryonic kidney 293 cell line was established that expressed an epitope-tagged δ-opioid receptor (DOR). Treatment of DOR cells with Tyr-d-Ala-Gly-Phe-d-Leu-enkephalin (DADL) resulted in a time-dependent decrease in the Bmax of δ-opioid receptor binding sites and immunoreactive receptor protein. When cells were coincubated with the proteasome inhibitor N-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal (ZLLL) and DADL, the magnitude of the agonist-induced decrease in Bmax and immunoreactive receptor protein was reduced compared with DADL treatment alone. Acute treatment of DOR cells with DADL caused a 3-fold increase in the level of phosphorylated mitogen-activated protein (MAP) kinase. Prior exposure of DOR cells to DADL completely abrogated the agonist-induced activation of MAP kinase. When DOR cells were coincubated with DADL and ZLLL, the proteasome inhibitor prevented the loss of agonist activation of MAP kinase. Acute treatment of DOR cell membranes with DADL stimulated [35S]guanosine 5′-3-O-(thio-)triphosphate (GTPγS) binding. When DOR cells were preincubated with DADL, the agonist-induced increase in [35S]GTPγS binding was attenuated. Coincubation of ZLLL and agonist partially prevented the decreased responsiveness to agonist stimulation. The results of this study demonstrated that inhibition of agonist-induced down regulation with a proteasome inhibitor attenuated opioid tolerance in a cellular model, and suggest that coadministration of a proteasome inhibitor with chronic opioid agonist treatment may be useful for limiting opioid tolerance in vivo.