BACKGROUND: Organophosphorus insecticides may promote the accumulation of acetylcholine at synapses and the neuromuscular junction by inhibiting acetylcholinesterase activity to cause disturbance of neural signal con...BACKGROUND: Organophosphorus insecticides may promote the accumulation of acetylcholine at synapses and the neuromuscular junction by inhibiting acetylcholinesterase activity to cause disturbance of neural signal conduction and induce a toxic reaction. Organophosphorus insecticides may act on M2 muscarinic acetylcholine receptors, whose combination with G proteins is regulated by phosphorylation of G protein-coupled receptor kinase 2. OBJECTIVE: To investigate the effects of organophosphorus insecticides on the phosphorylation of G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptors and to reveal other possible actions of organophosphorus insecticides. DESIGN, TIME AND SETTING: An observational study, which was performed in the Central Laboratory of Shenyang Medical College, and Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University from June 2002 to December 2004. MATERIALS: Paraoxon, parathion, chlorpyrifos, and chlorpyrifos oxon were provided by Chem Service Company, USA, [γ -p^32] ATP and [^35S]GTP γ S by New England Nuclear Life Science Products, and recombinant β 2-adrenergic receptor membrane protein by Sigma Company, USA. METHODS: The M2 muscarinic acetylcholine receptor was extracted and purified from pig brain using affinity chromatography. Subsequently, the purified M2 muscarinic acetylcholine receptor, G protein-coupled receptor kinase 2, and [γ -p^32] ATP were incubated with different concentrations of paraoxon and chlorpyrifos oxon together. The mixture then underwent polyacrylamide gel electrophoresis, and the gel film was dried and radioactively autographed to detect phosphorylation of the M2 muscarinic acetylcholine receptor. Finally, the radio-labeled phosphorylated M2 receptor protein band was excised for counting with an isotope liquid scintillation counter. MAIN OUTCOME MEASURES: Effects of chlorpyrifos oxon, paraoxon, chlorpyrifos, and parathion in different concentrations on the phosphorylation of the M2 muscarinic acetylcholine receptor; effects of chlorpyrifos oxon on the phosphorylation of the β -adrenergic receptor. RESULTS: Chlorpyrifos oxon could completely inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor, and its IC50 was 70 μ mol/L. Chlorpyrifos could also inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor. However, paraoxon and parathion could not inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor. Chlorpyrifos oxon in different concentrations could also not inhibit the phosphorylation of the β 2-adrenergic receptor catalyzed by G protein-coupled receptor kinase 2. CONCLUSION: Different kinds of organophosphorus insecticides have different effects on the phosphorylation of the G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptor. Organophosphorus insecticides possibly have different toxic effects.展开更多
文摘BACKGROUND: Organophosphorus insecticides may promote the accumulation of acetylcholine at synapses and the neuromuscular junction by inhibiting acetylcholinesterase activity to cause disturbance of neural signal conduction and induce a toxic reaction. Organophosphorus insecticides may act on M2 muscarinic acetylcholine receptors, whose combination with G proteins is regulated by phosphorylation of G protein-coupled receptor kinase 2. OBJECTIVE: To investigate the effects of organophosphorus insecticides on the phosphorylation of G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptors and to reveal other possible actions of organophosphorus insecticides. DESIGN, TIME AND SETTING: An observational study, which was performed in the Central Laboratory of Shenyang Medical College, and Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University from June 2002 to December 2004. MATERIALS: Paraoxon, parathion, chlorpyrifos, and chlorpyrifos oxon were provided by Chem Service Company, USA, [γ -p^32] ATP and [^35S]GTP γ S by New England Nuclear Life Science Products, and recombinant β 2-adrenergic receptor membrane protein by Sigma Company, USA. METHODS: The M2 muscarinic acetylcholine receptor was extracted and purified from pig brain using affinity chromatography. Subsequently, the purified M2 muscarinic acetylcholine receptor, G protein-coupled receptor kinase 2, and [γ -p^32] ATP were incubated with different concentrations of paraoxon and chlorpyrifos oxon together. The mixture then underwent polyacrylamide gel electrophoresis, and the gel film was dried and radioactively autographed to detect phosphorylation of the M2 muscarinic acetylcholine receptor. Finally, the radio-labeled phosphorylated M2 receptor protein band was excised for counting with an isotope liquid scintillation counter. MAIN OUTCOME MEASURES: Effects of chlorpyrifos oxon, paraoxon, chlorpyrifos, and parathion in different concentrations on the phosphorylation of the M2 muscarinic acetylcholine receptor; effects of chlorpyrifos oxon on the phosphorylation of the β -adrenergic receptor. RESULTS: Chlorpyrifos oxon could completely inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor, and its IC50 was 70 μ mol/L. Chlorpyrifos could also inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor. However, paraoxon and parathion could not inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor. Chlorpyrifos oxon in different concentrations could also not inhibit the phosphorylation of the β 2-adrenergic receptor catalyzed by G protein-coupled receptor kinase 2. CONCLUSION: Different kinds of organophosphorus insecticides have different effects on the phosphorylation of the G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptor. Organophosphorus insecticides possibly have different toxic effects.