I₁-imidazoline Receptor Mediates The Modulation To Morphine Dependence By Agmatine

Previous studies showed agmatine, an endogenous I1-imidazoline receptor ligand, prevents morphine dependence, but the mechanism remains unclear. Present study, therefore, is to directly demonstrate whether I1-imidazoline receptor participates in the modulation to morphine dependence by agmatine, and to discuss its molecular mechanism in signal transduction aspect.Firstly, u opioid receptor (MOR) and I1-imidazoline receptor (I1R) were stably co-expressed in CHO cells to establish the cell model (CHO-u/I1). The Bmax values were 1.83 + 0.13 pmol/mg protein for MOR in [3H]diprenorphine binding assay and 43.59 + 6.83 fmol/106 cells for I1R in [3H]clonidine binding assay, respectively. After chronic treatment of CHO-u and CHO-u/Ii with morphine (10 uM, 48h), naloxone induced cAMP overshoot and elevation of intracellular Ca2+ concentration, which suggested that the cell model of morphine dependence/withdrawal was generated. Agmatine at low concentrations (0.01-2.5 uM) concentration-dependently attenuated the development of cAMP overshoot through I1R, while an additional inhibitory effect of agmatine at high concentrations (5-100 uM) might be related to both activating I|R and blocking L-type voltage-gated calcium channels. However, after the development of morphine dependence, agmatine did not alter the expression of cAMP pathway supersensitivity either in CHO-u or in CHO-u/I1. Agmatine acting on I1R not only inhibited the development of Ca2+ signal pathway up-regulation, but also attenuated its expression to a certain extent. In addition, agmatine (1 and 3 uM) activating I1R inhibited the increases of both phosphorylations of CREB and ERK, and expression of c-Fos during withdrawal.In conclusion, the model system of co-expressing MOR and I1R in CHO cells is generated for the first time. Present researches provide the first direct evidence for the participation of I1R in the inhibitory effects of agmatine on morphine dependence. Its mechanism may be related that agmatine acting on I1R inhibits compensatory adaptations to cAMP pathway and Ca2+ signal pathway during the development of morphine dependence, and then alters gene expression through the integration of cAMPpathway and Ca2+ signal pathway. Agmatine-I1R system prefers inhibiting the development of morphine dependence to the expression.