胍丁胺-I1咪唑啉受体对μ-阿片受体内吞的影响及机制探讨

Modulation of agmatine on internalization of μ-opioid receptor by activation of I1 imidazoline receptor and its molecular mechanism

【目的】观察胍丁胺通过激活I1咪唑啉受体(I1R)对阿片预处理引起的μ-阿片受体(MOR)内吞的影响及可能的分子基础。【方法】以CHO-μ和CHO-μ/IRAS(imidazoline receptor antisera-selected protein)细胞作为研究对象,用[3H]diprenorphine结合实验方法,确定胍丁胺-I1R作用系统对MOR内吞的影响以及可能产生的分子基础。【结果】在正常CHO-μ和CHO-μ/IRAS细胞中,DAMGO([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin,1μmol/L)预处理两细胞30min后,MOR均发生内吞。胍丁胺(1nM～1μM)和DAMGO共同预处理两细胞30min,胍丁胺能浓度依赖性地抑制由DAMGO预处理引起的CHO-μ/IRAS细胞中MOR的内吞,而相同浓度胍丁胺对CHO-μ细胞中由DAMGO预处理引起的MOR的内吞无显著影响,且这一作用能被依法克生所阻断,提示胍丁胺通过激活I1R对DAMGO预处理引起的MOR内吞具有显著的抑制作用。Ser375磷酸化是影响MOR内吞的重要因素之一,而胍丁胺(10nM～1μM)和DAMGO共同预处理CHO-μ/IRAS和CHO-μ细胞30min,对两细胞中MORSer375磷酸化作用均无显著性影响,提示胍丁胺-I1R作用系统并不是通过抑制MORSer375磷酸化作用而抑制MOR内吞的。【结论】胍丁胺通过激活I1R可抑制DAMGO处理所致的μ-阿片受体内吞,此作用可能与胍丁胺抑制阿片依赖有关,但其分子机制并不是通过抑制MOR Ser375磷酸化作用而抑制MOR内吞。

[ Objective] To investigate the effects of agmatine by activation I1 imidazoline receptor on DAMGO-induced internalization of μ-opioid receptor. [ Methods] Two cell lines, Chinese hamster ovary cells expressing μ-opioid receptor alone （CHO-μ） and co-expressing μ-opioid receptor and imidazoline receptor antisera-selected protein （IRAS）, a candidate for I1 imidazoline receptor, （ CHO-μ/IRAS）, were used. [^3H] diprenorphine binding assay were used to determine the effect on the internalization of μ-opioid receptor by agnatine-I1 imidazoline receptor system and its molecular mechanism. [Results] In normal CHO-μ and CHO-μ/IRAS cells, treatment by DAMGO （ [D-Ala2, N-Me-Phe4, Gly5-ol] -enkephalin, 1 μM, 30 min） induced internalization of MOR in both cells. Agmatine （ 1 nM - 1 μM） concentration-dependently inhibited DAMGO-induced internalization of MOR in CHO-μ/IRAS cells, while this effect was not observed in CHO-μ cells. Efaroxan completely reversed the effect of agmatine in CHO-μ/IRAS cell. This result suggested that agmatine acting on I1R inhibits internalization of MOR. The Ser^375 phosphorylation of MOR is one of the important factor effecting the internalization of MOR. Agmatine （ 10 nM - 1 μM） did not alter the DAMGO-induced Ser^375 phosphorylation of MOR either in CHO-μ or in CHO-μ/RAS. These result indicated that Agmatine-I1R inhibited the ntemalization of MOR was not by inhibiting the Ser^375 phosphorylation of MOR. [ Conclusions] Agmatine could inhibit DAMGO-induced internalization of μ-opioid receptor by activation I1 imidazoline receptor. This action may contribute to the inhibition of agmatine to opioid dependence, but its mechanism may not be related that agmatine acting on phosphorylation of Ser^375 of μ-opioid receptor.