Chlorpyrifos oxon CPO activates extracellular signal-regulated kinase ERK 44/42 in Chinese hamster ovary CHOK1 cells but the mechanism is not defined This study tests the hypothesis that diacylglycerol DAG is the secondary messenger responsible for CPO-induced ERK 44/42 activation It is known that DAG is sequentially hydrolyzed by DAG lipase and monoacylglycerol MAG lipase both of which are organophosphate sensitive Inhibition of these enzymes might therefore lead to the accumulation of DAG and MAG of which only DAG is a secondary messenger The experiments show that treatment of CHOK1 cells with CPO significantly inhibits DAG/MAG lipase activity and elevates cellular DAG levels Pretreatment of CHOK1 cells with CPO or a carbamate known to be a DAG lipase inhibitor followed by treatment with a cell-permeable DAG 1,2-dihexanoyl-sn-glycerol results in synergistic activation of ERK 44/42 CPO-potentiated DAG-induced ERK 44/42 activation is both time and concentration dependent This activation is blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase suggesting that these enzymes are important in CPO/DAG cellular signaling Activation by a stable DAG analogue phorbol ester was not altered by CPO suggesting that DAG metabolism is the probable target for CPO-potentiated DAG-induced ERK 44/42 activation These observations support the hypothesis that CPO potentiates DAG signaling in CHOK1 cells by inhibiting a CPO-sensitive DAG lipase thereby providing a potential mechanism of toxicity not associated with acetylcholinesterase inhibition
Title: Diethylphosphorylation of rat cardiac M2 muscarinic receptor by chlorpyrifos oxon in vitro Bomser JA, Casida JE Ref: Toxicol Lett, 119:21, 2001 : PubMed
The acute toxicity of chlorpyrifos oxon (CPO), the metabolically-activated form of the major organophosphorus insecticide chlorpyrifos, is attributable to diethylphosphorylation of acetylcholinesterase at its esteratic site. As a secondary effect, CPO is known to compete with agonist binding to the M2 muscarinic acetylcholine receptor (mAChR). This study tests the hypothesis that [ethyl-1,2-(3)H]CPO labels the M2 mAChR in rat cardiac membrane proteins. Of four labeled protein regions observed, only one had an apparent molecular mass (70-75 kDa) consistent with that of glycosylated M2 mAChR. It was identified as M2 muscarinic receptor by Western blotting and immunoprecipitation using a cardiac-specific M2 mAChR monoclonal antibody, providing the first direct evidence for diethylphosphorylation of a muscarinic receptor. This may be a functionally important M2 mAChR site, but the toxicological relevance and species and organ specificity of diethylphosphorylation are unknown.
