某些有机磷杀虫剂影响乙酰胆碱M2受体磷酸化的实验研究

Effects of organophosphorus insecticides on G-protein coupled receptor kinase 2 mediated phosphorylation of m2 muscarinic receptors

目的研究有机磷类杀虫剂(OPs)对G蛋白结合受体激酶-2(GRK-2)介导的毒蕈碱乙酰胆碱m2受体(mAChR2)磷酸化的影响,进一步揭示OPs可能存在的其他作用途径。方法将纯化的大鼠脑mAChR2,GRK-2,同位素磷32标记的三磷酸腺苷([γ-P32]ATP)与不同浓度的对氧磷(PO)、氧毒死蜱(CPO)和毒死蜱(CPF)共同保温,聚丙烯酰胺凝胶电泳,凝胶片干燥后放射性自显影检测mAChR2磷酸化结果。将干燥凝胶片中放射性标记的磷酸化mAChR2蛋白带剪下,同位素液体闪烁计数器计数。结果CPO抑制大鼠脑mAChR2的磷酸化,其IC50为70?mol/L;CPF也抑制mAChR2的磷酸化,而PO与对硫磷(PT)不抑制mAChR2的磷酸化。CPO和CPF并不抑制同样由GRK2介导的β2肾上腺素受体(β2-AR)的磷酸化。结论CPO和CPF选择性地抑制GRK-2介导的mAChR2磷酸化,而PO和PT无此效应。说明某些有机磷类杀虫剂可能存在其它作用靶分子。

Objective To explore the effect of organophosphorus insecticides(OPs)on G-protein coupled receptor kinase 2-mediated phosphorylation of m2 muscarinic receptors in vitro and to understand an alternative target of the OPs for human and other animals.Methods The acetylcholine m2 muscarinic receptors purified from rat brain by single step affinity chromatography;In vitro experiments,the purified m2 receptor was incubated with varying concentrations of paraoxon,chlorpyrifos oxon or the parent insecticides along with G-protein coupled receptor kinase 2(GRK2)and [γ-p(32)] ATP.The proteins were separated by electrophoresis,gels were dried and autoradioprams developed.Bands containing m2 receptor were excised and counted by liquid scintillation.Results Chlorpyrifos oxon inhibited phosphorylation of m2 muscarinic receptors by GRK2 with a median inhibition concentration(IC50) of 70umol/L.Chlorpyrifos also inhibited m2 receptors phosphorylation,however it was less potent and less efficacious than that of chlorpyrifos oxon.Interestingly,paraoxon and parathion had little effect on the receptor phosphorylation under the same conditions.Conclusion Chlorpyrifos oxon and chlopyrifos may alter GRK2-mediated regulatory pathways for m2 receptors by direct interaction with m2 receptor.These differential actions could contribute to differences in toxicity following exposure to different organophosphorus toxicants.