Fig. 7.

Effect of administering morphine or DAMGO to mice on the stability of the MOR-HINT1 association in PAG synaptosomal plasma membranes. Immunoprecipitated MOR from murine PAG was labeled by antibodies directed to different peptide sequences: NT N terminal sequence (2–16: DSSAGPGNISDCSDP); 2EL Second External Loop (208–216: TKYRQGSID); CT C terminal sequence (387–398: LENLEAETAPLP); 3IL Third Internal Loop (256–269: ILRLKSVRMLSGSK). IP immunoprecipitation, WB Western blot. Deglycosylated MOR migrates at 38 kDa: WGL wheat germen lectin-purified glycosylated fraction of synaptosomal proteins from mouse cerebral cortex. HINT1 (93–106: GYRMVVNEGADGGG). RGSZ2: IQ internal sequence (46–60:EERGDSSGRSPHTTK); CT C terminal sequence (192–202: NSQIYKAFVES); W15 (sc-48286) internal region. Ψ The corresponding antibody (affinity-purified IgGs) was pre-incubated with 0.05 mg of the antigenic peptide for 1 h at room temperature before probing the proteins from PAG synaptosomes. Mice received 10 nmol morphine and 24 h later they were injected with 1 nmol Gö7874 (PKC inhibitor) or MK-801 (NMDAR antagonist). After 30 min, they were killed and the MOR present in the PAG was immunoprecipitated. The association of HINT1 and RGSZ2 with the MOR was then studied. Data are the mean ± SEM from three independent assays; the control mice received no morphine. *Significantly different from the control, ANOVA-Student–Newman–Keuls test; p < 0.05. Mice were icv-injected with 0.2 nmol DAMGO, a dose that produced a peak effect of about 80% of the maximum possible antinociception when evaluated in the warm water 52°C tail-flick test [35]. The mice were killed and the PAG was removed at the times indicated post-opioid injection. The MOR-HINT1 association was determined in the soluble fraction of synaptosomes, which contained the internalized and probably newly synthesized receptor [42]