Agmatine-I1 Imidazoline Receptor Mediates The Modulation To Compensatory Adaptation Of μ-opioid Receptor By Long-term Treatment Of Opioids

Previous studies showed agmatine, an endogenous I₁-imidazoline receptor ligand, prevents opioid dependence in the post-acceptor level, which may be related that agmatine acting on I1R inbibits compensatory up-regulation after long-term treatment of opioid. Is agmatine preventing opioid dependence in the acceptor level relate to opioid acceptor compensatory change through activating I1R? There are no reports about this. Present study, therefore, is to directly demonstrate whether agmatine-I1R system participates in the modulation to opioid acceptor compensatory adaptation in the acceptor level after long-term treatment of opioid, and to discuss its molecular mechanism.Firstly, Two cell lines, Chinese hamster ovary cells expressingμ-opioid receptor alone (CHO-μ) and co-expressingμ-opioid receptor and imidazoline receptor antisera-selected protein (IRAS), a candidate for I1 imidazoline receptor, (CHO-μ/IRAS), were used to show the relationship the opioid function regulation of agmatine and I1R. There was no significant difference of expression of MOR and affinity to ligand as well as the reaction to agonist between normal CHO-μand CHO-μ/IRAS cells. DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin, 10μM)treatment for 30 min induced desensitization of MOR in CHO-μ/IRAS cells, while agmatine (10nM～10μM) did not influence DAMGO-induced desensitization ofμ-opioid receptor. Chronic treatment by DAMGO (1μM, 12 h) decreased the expression of MOR in CHO-μand CHO-μ/TRAS cells. Agmatine (1～100nM) concentration-dependently inhibited DAMGO-induced down-regulation of MOR in CHO-μ/IRAS cells, while this effect was not observed in CHO-μcells. Efaroxan, an I1 imidazoline receptor-preferential antagonist, completely reversed the effect of agmatine in CHO-μ/IRAS cell. DAMGO (1μM) treatment for 30 min induced internalization of MOR in both cells. Agmatine (1nM～1μM) concentration-dependently inhibited DAMGO-induced intemalization of MOR in CHO-μ/IRAS cells, while this effect was not observed in CHO-μcells. Efaroxan completely reversed the effect of agmatine in CHO-μ/IRAS cell. The Ser³⁷⁵ phosphorylation of MOR was significantly increased after DAMGO (5μM) treatment for 30 min in both cells. This result suggested that the Ser³⁷⁵ phosphorylation of MOR is one of the important factor effecting the intemalization of MOR. Agmatine (10nM～1μM ) did not alter the DAMGO-induced Ser³⁷⁵ phosphorylation of MOR either in CHO-μor in CHO-μ/IRAS. These result indicated that Agmatine-I1R inhibited the intemalization of MOR was not by inhibiting the Ser³⁷⁵ phosphorylation of MOR. Meanwhile, Agmatine (10 nM～1μM) did not influence the expression of MOR mRNA in CHO-μ/IRAS cell.In conclusion, the model system of co-expressing MOR and I1R in CHO cells is used for the first time. Present researches provide the first direct evidence for the participation of I1R in the inhibitory effects of agmatine on opioid tolerance and dependence in the receptor level. Its mechanism may be related that agmatine acting on I1R inhibits intemalization of MOR, and then inhibits its downregulation. Agmatine-I1R system has no influence on the expression of MOR and phosphorylation of Ser³⁷⁵ in the process of inhibiting opioid tolerance and dependence.