Udarbe Zamora Elmar M, Liu Jing, Pope Carey N
Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
J Toxicol Environ Health A. 2008;71(21):1440-7. doi: 10.1080/15287390802328887.
The muscarinic M2 receptor is a member of the G protein-coupled receptor (GPCR) superfamily. Agonist activation of GPCR leads to their phosphorylation, desensitization, internalization, and subsequent endocytic recycling or lysosomal degradation. Agonist-induced phosphorylation of M2 receptors is mediated by G-protein receptor kinase 2 (GRK2). The active metabolite of the organophosphorus insecticide chlorpyrifos, i.e., chlorpyrifos oxon (CPO), inhibited agonist-induced phosphorylation of human recombinant M2 receptors by GRK2 in vitro in a concentration-dependent manner. In both intact HEL 299 cells (human embryonic lung fibroblasts expressing M2 receptors) and CHO-M2 cells (stably expressing M2 receptors), the muscarinic agonist carbachol (100 microM) led to receptor internalization as determined by reduced specific binding to the membrane-impermeable radioligand [(3)H]-N-methylscopolamine (NMS). CPO alone (100 microM) exerted no significant effect on NMS binding in either HEL 299 or CHO-M2 cells. In HEL 299 cells, CPO did not influence carbachol-induced internalization, whereas in CHO-M2 cells CPO blocked internalization. In primary striatal neurons, M2 receptors appeared widely and diffusely distributed. Exposure to either carbachol or CPO led to apparent receptor internalization with an increased percent of cells exhibiting punctate domains of immunostaining, while combined exposure to both carbachol and CPO led to a significantly higher percent of cells exhibiting this appearance. The data suggest that CPO may differentially influence agonist-stimulated M2 receptor internalization in a cell-dependent manner.
毒蕈碱型M2受体是G蛋白偶联受体（GPCR）超家族的成员。GPCR的激动剂激活会导致其磷酸化、脱敏、内化，以及随后的内吞再循环或溶酶体降解。毒蕈碱型M2受体的激动剂诱导的磷酸化由G蛋白受体激酶2（GRK2）介导。有机磷杀虫剂毒死蜱的活性代谢物，即毒死蜱氧磷（CPO），在体外以浓度依赖性方式抑制GRK2对人重组M2受体的激动剂诱导的磷酸化。在完整的HEL 299细胞（表达M2受体的人胚胎肺成纤维细胞）和CHO-M2细胞（稳定表达M2受体）中，毒蕈碱激动剂卡巴胆碱（100 microM）导致受体内化，这通过与膜不透性放射性配体[（3）H]-N-甲基东莨菪碱（NMS）的特异性结合减少来确定。单独的CPO（100 microM）对HEL 299或CHO-M2细胞中的NMS结合没有显著影响。在HEL 299细胞中，CPO不影响卡巴胆碱诱导的内化，而在CHO-M2细胞中CPO阻断内化。在原代纹状体神经元中，M2受体广泛且分散分布。暴露于卡巴胆碱或CPO都会导致明显的受体内化，表现为免疫染色呈点状结构域的细胞百分比增加，而同时暴露于卡巴胆碱和CPO会导致表现出这种外观的细胞百分比显著更高。数据表明，CPO可能以细胞依赖性方式差异性地影响激动剂刺激的M2受体内化。
