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 irreversi ble 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 para oxon toxicity developed rapidly. The hypothesis was tested that change s in the kinetics of reactivity of AChE with its substrate acetylcholi ne (ACh) and the inhibitor paraoxon contribute to the observed toleran ce, The kinetic constants V-max and K-m were determined by Lineweaver- Burk transformations. The affinity (K-d), phosphorylation (k(p)) and t he bimolecular rate (k(i)) constants were established from slopes and standard deviations of inhibition curves. Acetylcholinesterase propert ies of brain and diaphragm from controls and paraoxon-tolerant rats we re compared, In controls, K-m, determining the affinity of AChE for AC h, 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 K-m for diaphragm was reduced to 0.047 x 10(-3) M and the K-m for brain to 0.057 x 10(-3) M. This decrease was seen with all paraoxon concentrations and was significantly different from cont rols 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 trea tment with paraoxon caused a fivefold decrease in the dissociation con stant (K-d) from 1.56 to 0.268 mu M and a threefold decrease in the ph osphorylation constant (k(p)) 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 rema ining reduced amount of AChE can hydrolyze ACh more efficiently, regar dless of the change in sensitivity to the inhibitor, The observed chan ges may be the result of structural changes of AChE or the result of a ltered levels of preexisting isozymes of AChE. (C) 1996 Academic Press , Inc.