Received for publication September 8, 2006.
Revised December 6, 2006.
Accepted for publication December 8, 2006.

Inhibition of agonist-induced down regulation of the opioid receptor with a proteasome inhibitor attenuates opioid tolerance in human embryonic kidney 293 cells

Abstract

This study was designed to test the hypothesis that inhibition of agonist-induced receptor down regulation would block the development of opioid tolerance in a cell-based model. A HEK293 cell line (DOR) was established that expressed an epitope-tagged opioid receptor. Treatment of DOR cells with Tyr-D-Ala-Gly-Phe-D-Leu (DADL) resulted in a time-dependent decrease in the B_max of opioid receptor binding sites and immunoreactive receptor protein. When cells were co-incubated with the proteasome inhibitor, N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal (ZLLL), and DADL, the magnitude of the agonist-induced decrease in B_max 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 co-incubated 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]GTPS binding. When DOR cells were preincubated with DADL, the agonist-induced increase in [³⁵S]GTPS binding was attenuated. Co-incubation 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 co-administration of a proteasome inhibitor with chronic opioid agonist treatment may be useful for limiting opioid tolerance in vivo.

Key words: G protein coupled receptor, Map kinase, receptor, opioid, proteasome inhibitor, tolerance