Teixeira Simone A, Castro Gláucia M, Papes Fábio, Martins Maria L, Rogério Fábio, Langone Francesco, Santos Leonilda M B, Arruda Paulo, de Nucci Gilberto, Muscará Marcelo N
Department of Biochemistry, UNICAMP, Campinas, SP, Brazil.
Brain Res Mol Brain Res. 2002 Feb 28;99(1):17-25. doi: 10.1016/s0169-328x(01)00341-2.
The activity and expression of nitric oxide synthase (NOS) isoforms and protein nitrotyrosine (NT) residues were investigated in whole encephalic mass (WEM) homogenates during the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. EAE stages (0-III) were daily defined by clinical evaluation, and in the end of each stage, WEMs were removed for analysis of NOS activity, protein NT residues and mRNA for the different NOS isoforms. In the presence of NADPH, WEMs from EAE-III rats showed lower Ca2+-dependent NOS activity than those from control group. These differences disappeared in the presence of exogenous calmodulin, flavin adenine dinucleotide (FAD), tetrahydrobiopterin (BH4) and NADPH. Of all the cofactors, just the omission of FAD caused comparable decrease of Ca2+-dependent NOS activity from both groups. Ca2+-independent NOS activity from EAE-III animals was insensitive to the omission of any of the cofactors, while in control animals this activity was significantly inhibited by the omission of either FAD or BH4. Increased levels of both iNOS mRNA and protein NT expression were observed in animals with EAE, which also showed lower levels of a thermolabile NOS inhibitor in WEM homogenates and sera than controls. In conclusion, during late EAE stages, constitutive Ca2+-dependent NOS activity decreases concomitantly with iNOS upregulation, which could be responsible for the high protein NT levels. The differential dependence of iNOS activity on cofactors and the absence of an endogenous thermolabile NOS inhibitor in animals with EAE could reflect additional control mechanisms of NOS activity in this model of multiple sclerosis.
在Lewis大鼠实验性自身免疫性脑脊髓炎（EAE）发展过程中，对全脑匀浆（WEM）中一氧化氮合酶（NOS）亚型的活性和表达以及蛋白质硝基酪氨酸（NT）残基进行了研究。通过临床评估每日确定EAE阶段（0 - III），在每个阶段结束时，取出WEM用于分析不同NOS亚型的NOS活性、蛋白质NT残基和mRNA。在存在NADPH的情况下，EAE - III期大鼠的WEM显示出比对照组更低的Ca2 +依赖性NOS活性。在存在外源性钙调蛋白、黄素腺嘌呤二核苷酸（FAD）、四氢生物蝶呤（BH4）和NADPH时，这些差异消失。在所有辅助因子中，仅省略FAD会导致两组的Ca2 +依赖性NOS活性出现类似程度的降低。EAE - III期动物的Ca2 +非依赖性NOS活性对任何辅助因子的省略均不敏感，而在对照动物中，省略FAD或BH4会显著抑制该活性。在患有EAE的动物中观察到诱导型NOS（iNOS）mRNA和蛋白质NT表达水平升高，这些动物的WEM匀浆和血清中热不稳定NOS抑制剂的水平也低于对照组。总之，在EAE后期阶段，组成型Ca2 +依赖性NOS活性与iNOS上调同时降低，这可能是蛋白质NT水平升高的原因。EAE动物中iNOS活性对辅助因子的不同依赖性以及内源性热不稳定NOS抑制剂的缺失可能反映了该多发性硬化模型中NOS活性的额外调控机制。
