Lapadula E S, Lapadula D M, Abou-Donia M B
Department of Pharmacology, Duke University Medical Center, Durham, NC 27710.
Neurochem Int. 1992 Feb;20(2):247-55. doi: 10.1016/0197-0186(92)90174-p.
Calcium- and calmodulin-regulated protein phosphorylation has been suggested to play a role in the pathogenesis of organophosphorus compound-induced delayed neurotoxicity (OPIDN). This condition is characterized by ataxia that progresses to paralysis concurrent with a central-peripheral distal axonopathy after a delay period of 1-2 weeks following exposure to an organophosphorus compound causing delayed neurotoxicity, such as tri-o-cresyl phosphate (TOCP). Calcium/calmodulin (CaM) kinase II is involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing OPIDN. In this study, chickens were given a single oral neurotoxic dose of 750 mg TOCP/kg body weight and killed after 1, 6, 14 or 21 days following treatment. Protein kinase-mediated phosphorylation of cytoskeletal proteins was studied in proximal and distal parts of sciatic nerves of control and treated hens. Peripheral nerve proteins were phosphorylated in vitro using [gamma-32P]ATP as a phosphoryl group donor. Phosphorylated proteins were separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein phosphorylation was detected by autoradiography and quantified by laser microdensitometry. The extent of Ca2+-calmodulin dependent phosphorylation of five cytoskeletal proteins was significantly increased in TOCP treated animals, particularly at 1 and 6 days after treatment, in both the proximal and distal portion of the nerve. The identity of these proteins was confirmed by 2-D PAGE as tubulin, the neurofilament triplet proteins and microtubule associated protein-2 (MAP-2). These results confirm earlier observation of the close temporal relationship between increased cytoskeletal protein phosphorylation and the development and OPIDN.
钙和钙调蛋白调节的蛋白质磷酸化被认为在有机磷化合物诱导的迟发性神经毒性（OPIDN）的发病机制中起作用。这种病症的特征是共济失调，在接触导致迟发性神经毒性的有机磷化合物（如三邻甲苯基磷酸酯（TOCP））后1-2周的延迟期后，共济失调进展为瘫痪，并伴有中枢-外周远端轴索病。钙/钙调蛋白（CaM）激酶II参与在用能够产生OPIDN的有机磷化合物处理动物后，脑微管和脊髓神经丝三联体蛋白磷酸化的增加。在本研究中，给鸡口服单次神经毒性剂量750 mg TOCP/kg体重，并在处理后1、6、14或21天处死。研究了对照母鸡和处理后母鸡坐骨神经近端和远端部分中蛋白激酶介导的细胞骨架蛋白磷酸化。使用[γ-32P]ATP作为磷酰基供体在体外对周围神经蛋白进行磷酸化。通过一维和二维十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离磷酸化蛋白。通过放射自显影检测蛋白磷酸化，并通过激光显微密度测定法定量。在TOCP处理的动物中，特别是在处理后1天和6天，神经近端和远端部分中五种细胞骨架蛋白的Ca2 + -钙调蛋白依赖性磷酸化程度显著增加。通过二维聚丙烯酰胺凝胶电泳确认这些蛋白为微管蛋白、神经丝三联体蛋白和微管相关蛋白-2（MAP-2）。这些结果证实了早期观察到的细胞骨架蛋白磷酸化增加与OPIDN的发生之间的密切时间关系。
