| Opiate abuse is a serious problem which has huge impact on economic prosperity, population quality and social stability. Thus, gaining a deep insight into the neurobiological mechanisms of opioid dependence and discovering the anti-relapsing drugs are not only of great scientific significance, but also of important social and economic value.Agmatine is a novel neurotransmitter and considered as an endogenous ligand for imidazoline receptors. Our previous study found agmatine could inhibit opioid tolerance, physical and psychological dependence. Though previous studies suggested that I1-imidazoline receptor (I1R) may be involved in the mechanisms of agmatine on regulating opioid functions, whether I1R is the target of agmatine remains unclear. The reason is that agmatine could target several molecules which have close relations with opioid dependence. On the other hand, selective antagonists for I1R are still unavailable. In 2000, imidazoline receptor antisera-selected protein (IRAS), the I1R candidate protein, was cloned and proved to be the wild functional I1R. In this study, therefore, whether I1R is the target of agmatine modulating opioid dependence was investigated by RNA interference (RNAi) in vitro and in vivo to down-regulate the expression of I1R.In vitro, co-transfectedμopioid receptor and IRAS cell line (CHO-μ/IRAS cell) was used to study whether I1R is the target of agmatine in inhibiting-opioid dependence. LipofectamineTM2000 was used to transfect siRNA into CHO-μ/IRAS cells and the effective siRNA was screened by RT-PCR. In the experiments on the time-dependent and concentration-dependent effect of siRNA, we found that the expression of IRAS could still be down-regulated even when the concentration of siRNA was 40 nM, and that effect was most obvious after siRNA transfected for 24h at mRNA level and 48 h at protein level, respectively. This down-regulation effect could persist for about 2 days. After the down-regulation effect of siRNA was confirmed, we studied the function of agmatine on opioid dependence and its relationship with I1R by adenosine 3',5'-cyclic phosphate (cAMP) overshoot, a received marker to reflect the dependence and withdrawal state in vitro, which is induced by morphine chronic treatment and naloxone precipitation. cAMP overshoot was measured by a competitive immuno-fluorescence method (LANCETM cAMP384 kit). After CHO-μ/IRAS cell was treated by morphine (100μM) for 24 h and precipitated by naloxone (100μM), cAMP level was increased to 3 fold compared with that of vehicle control, indicating morphine dependence developed in this cell line. Co-pretreatment of CHO-μ/IRAS cells with agmatine (10 nM-10μM) during morphine exposure decreased cAMP overshoot in a concentration-dependent manner, and the IC50 value of inhibiting cAMP overshoot by agmatine was 0.36μM. Down-regulation of I1R expression by siRNA was used to study the relationship between I1R and the effect of agmatine. During the period of I1R down-regulating at protein level (48 h-72 h after siRNA transfected), CHO-μ/IRAS cells were treated with morphine and/or agmatine for 24 h. Compared with ns-siRNA transfected or vehicle treated cells, the inhibitory effect of agmatine (100 nM and 1μM) on cAMP overshoot was significantly reduced in siRNA transfected cells, indicating that I1R mediated the effect of agmatine on morphine dependence.Because of the difference between the transfected cell line (CHO-μ/IRAS cells) and the neuron expressing endogenous I1R and opioid receptors, we further studied the relationship between I1R and the effect of agmatine on opioid dependence in primary cultured hippocampal neurons of newborn rat. TransMessenger TM was used to transfect siRNA into hippocampal neurons and RT-PCR was used to screen the effective siRNA. In the experiments on the time-dependent effect by siRNA, we found that I1R was significantly down-regulated in hippocampal neurons after siRNA transfected for 24 h at mRNA level, and 72 h at protein level, respectively. This down-regulation effect could persist at least for 3 days. We proposed that siRNA complex may be more stable in hippocampal neurons and hippocampal neurons have no division and proliferation, which resulted in the difference of the time-courses by siRNA between hippocampal neurons and CHO-μ/IRAS cells.We firstly established the morphine dependence model in hippocampal neurons before investigating the effect of agmatine on morphine dependence. We found that after hippocampal neurons treated with morphine (10μM) for 24 h and then precipitated by naloxone (10μM), cAMP overshoot was not induced. However, when the time of morphine treatment was extended to 48 h, then precipitated by naloxone, the cAMP level was increased to about 2.7 fold compared with that of vehicle control, indicating that morphine dependence developed in the hippocampal neurons. Treatment with agmatine alone for 48 h had no effect on cAMP level, while co-administration of agmatine and morphine could significantly inhibit naloxone-precipitated cAMP overshoot in morphine-dependent neurons, the IC50 was 3.4μM. From the above results, we could conclude that agmatine inhibited morphine dependence in cultured primary hippocampal neurons. In order to study whether I1R is involved in the inhibitory effect of agmatine on morphine dependence, efaroxan, the imidazoline receptor/α2-adrenergic receptor (α2-AR)-mixed antagonist, and yohimbine, theα2-AR antagonist, were used. We found that pretreatment of efaroxan (10μM, 48 h) or yohimbine (10μM, 48 h) had no effect on basal cAMP level or naloxone-induced cAMP overshoot when given alone or with morphine. However, efaroxan, rather than yohimbine, could attenuate the inhibitory effect of agmatine (100 nM, 1μM) on naloxone-induced cAMP overshoot in morphine-dependent neurons, indicating that I1R may participate in the effect of agmatine. Down-regulating I1R expression in hippocampal neurons by siRNA was used to further study the relationship between I1R and the effect of agmatine. During the period of I1R down-regulated at protein level (48 h-96 h after siRNA transfected), hippocampal neurons were treated with morphine and/or agmatine for 48 h. Results showed that the inhibitory effect of agmatine (100 nM and 1μM) on morphine-treated, naloxone-precipitated cAMP overshoot was significantly reduced in siRNA transfected cells, compared with ns-siRNA transfected cells and vehicle treated cells, indicating that I1R mediated the inhibitory effect of agmatine on morphine dependence in primary cultured hippocampal neurons.Based on the above study in vitro, the role of I1R in agmatine-inhibiting morphine dependence by RNAi in vivo was conducted. Lipid carrier (DOPE + jetSITM10 mM) for mice brain was used to mediate siRNA into the lateral ventricle and the near brain regions of the mouse in vivo. Stereotaxic apparatus and guide cannulas were used for chronic injection of siRNA complex. We found that the mouse I1R at mRNA level or protein level was significantly reduced after 5 days or 7 days of continuously injection of siRNA complex, respectively. With the increase of time of siRNA treatment in vivo, the down-regulating effect became more obvious.In the study of the relationship between I1R and the inhibitory effect of agmatine on morphine dependence, two experiments were performed. One was to observe naloxone-precipitated withdrawal signs of the morphine dependent mice, and the other was to measure FosB andΔFosB expression in striatum and nucleus accumbens (NAc). After the mice were treated by siRNA, ns-siRNA or vehicle for 7 days, morphine (30 mg/kg,s.c) and/or agmatine (5 mg/kg,s.c.) were injected three times daily for 4 days. On day 5, all mice received naloxone (20 mg/kg, i.p.) to precipitate morphine withdrawal syndrome. In vehicle, ns-siRNA pretreated or siRNA pretreated groups, mice treated with morphine alone displayed severe withdrawal syndrome, such as jumping, writhing, ptosis, teeth chattering, diarrhea and defecation. These signs were significantly attenuated in the mice co-treated with agmatine and morphine in ns-siRNA or vehicle control group. The results were consistent with our previous study, confirming the inhibitory effect of agmatine on morphine dependence. In siRNA pretreated group, however, the withdrawal signs in the mice co-treated with agmatine and morphine were not significantly attenuated compared with the mice treated with morphine alone, indicating that the inhibitory effect of agmatine on morphine dependence was reversed by siRNA pretreatment. The inhibitory percentage of agmatine on jumping, the most significant withdrawal sign, was 63.1±3.2% in vehicle control and 58.9±3.9% in ns-siRNA control, while 30.6±6.7% in siRNA group compared with morphine treated mice, indicating that down-regulating of I1R led to the reduced effect of agmatine on morphine dependence.Striatum and NAc were dissected from mice brains after the withdrawal syndrome was observed, then FosB andΔFosB expression were measured by Western blot. We found that FosB andΔFosB were increased in the mice of chronic morphine treatment followed by naloxone precipitation, andΔFosB increased more than FosB. In ns-siRNA or vehicle control group, agmatine co-treated with morphine significantly reduced the elevation of FosB andΔFosB expression induced by chronic morphine treatment and naloxone precipitation, while agmatine's inhibitory effect on FosB andΔFosB expression was reversed in siRNA treated group. SinceΔFosB is related to psychological dependence, these results indicated that I1R not only mediated the inhibitory effect of agmatine on morphine physical dependence, but also on morphine psychological dependence.In the present study, we studied the role of I1R in the inhibitory effect of agmatine on morphine dependence in vitro and in vivo by down-regulating the expression of I1R. We found that agmatine inhibited morphine dependence via the activation of I1R in both CHO-μ/IRAS cells and primary cultured hippocampal neurons. Furthermore, agmatine inhibited the withdrawal syndromes and the elevation of FosB andΔFosB in striatum and NAc in morphine-dependent and naloxone precipitated mice via the activation of I1R. Therefore, our study demonstrated that I1R was the main target of agmatine on inhibiting morphine dependence for the first time, and further validated that agmatine and imidazoline receptors may constitute a novel system of modulating opioid functions.
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