Effect of agmatine on DAMGO-induced mu-opioid receptor down-regulation and internalization via activation of IRAS, a candidate for imidazoline I 1 receptor

Abstract

Agmatine, an endogenous ligand for imidazoline I 1 receptor, has previously been shown to prevent opioid tolerance in rats and mice, but the cellular mechanisms remain unknown. In the present study, the effects of agmatine activation on imidazoline I 1 receptor on the desensitization, down-regulation and internalization of μ opioid receptor were investigated. Two cell lines, CHO cells transfected μ opioid receptor (CHO-μ cells) and co-transfected μ opioid receptor and imidazoline I 1 receptor antisera-selected protein (IRAS) (CHO-μ/IRAS cells), were used. In both CHO-μ cells and CHO-μ/IRAS cells, agmatine (0.01–10 μM) did not affect the desensitization of μ opioid receptor induced by [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]-enkephalin (DAMGO) (10 μM) treatment for 30 min. However, agmatine (0.1–100 nM) co-pretreatment with DAMGO (1 μM) for 12 h concentration-dependently inhibited DAMGO-induced down-regulation of μ opioid receptor in CHO-μ/IRAS cells, but not in CHO-μ cells. Efaroxan, the I 1/α 2-adrenoceptors mix antagonist, completely reversed the inhibitory effect of agmatine, suggesting the participation of imidazoline I 1 receptor. In addition, agmatine (1–100 nM) inhibited DAMGO-induced internalization of μ opioid receptor in CHO-μ/IRAS cells, which was reversed by efaroxan as well. While treatment with DAMGO (1 μM) or co-treatment with agmatine (1–100 nM) for 12 h failed to affect the mRNA level of μ opioid receptor. Taken together, these results indicate that the inhibitory effect of agmatine on tolerance in vitro might be related to attenuation of the internalization and down-regulation of μ opioid receptor via activation of imidazoline I 1 receptor.