Milatovic D, Dettbarn W D
Department of Pharmacology, Vanderbilt University Medical School, Nashville, Tennessee 37212, USA.
Toxicol Appl Pharmacol. 1996 Jan;136(1):20-8. doi: 10.1006/taap.1996.0003.
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.
这些实验研究了暴露于对氧磷（一种乙酰胆碱酯酶的不可逆抑制剂）的大鼠在耐受性形成过程中乙酰胆碱酯酶（AChE）的变化。大鼠皮下注射0.09、0.12或0.19mg/kg的对氧磷，持续20天。对氧磷毒性临床症状的耐受性迅速形成。我们检验了这样一个假设，即AChE与其底物乙酰胆碱（ACh）及抑制剂对氧磷反应动力学的变化导致了所观察到的耐受性。通过Lineweaver-Burk转换确定动力学常数Vmax和Km。从抑制曲线的斜率和标准差确定亲和力（Kd）、磷酸化（kp）和双分子速率（ki）常数。比较了对照组和对氧磷耐受大鼠脑和膈肌的乙酰胆碱酯酶特性。在对照组中，决定AChE对ACh亲和力的Km，膈肌为0.063×10⁻³M，脑为0.072×10⁻³M。在对氧磷耐受大鼠中，AChE对ACh的亲和力增加，因为膈肌的Km降至0.047×10⁻³M，脑的Km降至0.057×10⁻³M。所有对氧磷浓度下均出现这种降低，且在治疗第5天后与对照组有显著差异。在膈肌（从27.30至45.14nM）和脑（从13.67至15.38nM）中观察到对氧磷的IC50值有小幅度但显著的增加。在脑中，用对氧磷进行20天治疗导致解离常数（Kd）从1.56降至0.268μM，降低了五倍，磷酸化常数（kp）从4.72降至1.52min⁻¹，降低了三倍。在膈肌中观察到的变化较小且不显著。对ACh亲和力的增加使耐受大鼠具有优势，因为剩余减少量的AChE能够更有效地水解ACh，而与对抑制剂敏感性的变化无关。所观察到的变化可能是AChE结构变化的结果，或者是AChE预先存在的同工酶水平改变的结果。
