Figure 2.

Upregulation of μ-opioid receptor expression during chronic morphine treatment. A, Immunoprecipitation of μ-opioid receptors from cell-lysate free samples (no lysate) and brain homogenates prepared from MOR^+/+ or MOR^−/− mice. Homogenates were prepared from entire brain after removal of the cerebellum. μ-Opioid receptors were immunoprecipitated with UMB-3 bound to protein A agarose beads. Samples were separated on 8% SDS-polyacrylamide gels and blotted. Blots were incubated with guinea pig anti-MOR antiserum {GP6}. Incubations were performed in the absence (−) or presence (+) of the peptide antigen. Note that UMB-3 selectively detects μ-opioid receptors and does not cross-react with other proteins present in tissue extracts prepared from MOR^−/− mice. B, MOR^+/+ mice were treated for 9 d with saline (lanes 1–4), escalating doses of morphine (10–90 mg/kg, s.c.) (lanes 5–9), or escalating doses of etonitazene (10–90 μg/kg, s.c.) (lanes 10–13). One hour after the last injection, brains were dissected. Homogenates were prepared from entire brain after removal of the cerebellum. μ-Opioid receptors were immunoprecipitated with UMB-3 and immunoblotted with guinea pig anti-MOR antiserum {GP6} (top). After UMB-3 immunoprecipitation, lysates were subsequently incubated with wheat germ lectin agarose beads to enrich glycoproteins. These samples were then probed with an anti-TFR antibody to confirm that equal amounts of lysate were used for UMB-3 immunoprecipitation (bottom). Each lane represents one animal. C, Blots were quantified by densitometry and expressed as percentage of μ-opioid receptor expression in saline-treated animals. Data correspond to the mean ± SEM. Results were analyzed by one-way ANOVA followed by the Bonferroni post hoc test (*p < 0.05). Note that μ-opioid receptor expression is upregulated during chronic morphine but not during chronic etonitazene. The positions of the molecular mass markers are indicated on the left (in kilodaltons).