Modification of acetylcholinesterase during adaptation to chronic, subacute paraoxon application in rat.

These experiments examined the changes in acetylcholinesterase (AChE) during tolerance development in rats exposed to paraoxon, an irreversible inhibitor of AChE. Rats were injected sc for 20 days with 0.09, 0.12, or 0.19 mg/kg of paraoxon. Tolerance to the clinical signs of paraoxon toxicity developed rapidly. The hypothesis was tested that changes in the kinetics of reactivity of AChE with its substrate acetylcholine (ACh) and the inhibitor paraoxon contribute to the observed tolerance. The kinetic constants Vmax and Km were determined by Lineweaver-Burk transformations. The affinity (Kd), phosphorylation (kp) and the bimolecular rate (ki) constants were established from slopes and standard deviations of inhibition curves. Acetylcholinesterase properties of brain and diaphragm from controls and paraoxon-tolerant rats were compared. In controls, Km, determining the affinity of AChE for ACh, was 0.063 x 10(-3) M and 0.072 x 10(-3) M for diaphragm and brain, respectively. In paraoxon-tolerant rats, the affinity of AChE for ACh increased since the Km for diaphragm was reduced to 0.047 x 10(-3) M and the Km for brain to 0.057 x 10(-3) M. This decrease was seen with all paraoxon concentrations and was significantly different from controls after the fifth day of treatment. Small, significant increases of IC50 values for paraoxon were observed in diaphragm (from 27.30 to 45.14 nM) and in brain (from 13.67 to 15.38 nM). In brain, a 20-day treatment with paraoxon caused a fivefold decrease in the dissociation constant (Kd) from 1.56 to 0.268 microM and a threefold decrease in the phosphorylation constant (kp) from 4.72 to 1.52 min-1. The observed changes in diaphragm were smaller and not significant. The increase in affinity to ACh gives an advantage to tolerant rats, because the remaining reduced amount of AChE can hydrolyze ACh more efficiently, regardless of the change in sensitivity to the inhibitor. The observed changes may be the result of structural changes of AChE or the result of altered levels of preexisting isozymes of AChE.