Participação do oxido nitrico no desenvolvimento da encefalomielite alergica experimental em ratos

AUTOR(ES)
DATA DE PUBLICAÇÃO

2001

RESUMO

Nitric oxide (NO) is a free radical endogenously synthetised by oxidation of one of the guanidinum nitrogen atoms of the aminoacid L-arginine by molecular oxygen. This biosynthesis is catalised by a family of enzimes generically known as nitric oxide synthases (NOS), which can be inhibited by structural analogs of L-arginine having substituent groups at the terminal guanidinum moeity. NO is extremely reactive toward different chemical species such as oxyigen, superoxide anion, thiol groups, transition metal ions, etc; nitrite (NO2-) and nitrate (NO3-) anions are the stable end products which result from NO oxidation within the physiological environment. NO, and nitrogen-derived reactive species (NDRS) as a whole, take part of several physiological processes in the brain, including neurotransmision, neuromodulation and synaptic plasticity, as well as pathological processes such as neurodegenerative diseases and neuroinflammation. Increased nitric oxide (NO) production has been associated with disease activity in experimental allergic encephalomyelitis (EAE). In this study, we characterized the expression and activity of the different NOS isoforms, as well as the presence of protein nitrotyrosine residues (NT, as a marker of peroxynitrite production) in whole encephalic mass homogenates during the development of EAE in rats. EAE was induced in Lewis rats by subcutaneous injection of an emulsion containing myelin basic protein and the development of the disease (stages Q - 111) was clinically evaluated daily. In the end of the different stages, whole encephalic masses (WEM) were removed for further analysis of NOS activity, protein NT residues (Western blot) and mRNA for the different NOS isoforms (RT-PCR). Blood samples were also collected for measurement of serum NO2- and NO3- as an index of systemic NO production. In the absence of the addition of any exogenous NOS cofactor (with the exception made for NADPH), Ca2+-dependent NOS activity was significantly decreased in rats with EAE at stage 111 when compared with either control, EAE-Q ar EAE-I groups, but no differences in Ca2+-independent NOS activity were observed among the groups. In the other hand, when the cofactors calmodulin (CaM), FAD, tetrahydrobiopterin (BH4) and NADPH were exogenously added, no differences in WEM Ca2+-dependent NOS activity were observed between control and EAE III groups. Similar decreases in Ca2+dependent NOS from either control or EAE 111 rats were observed when FAD was omitted from the incubation media, but no changes occurred in the absence of the other cofactors. A non-significant increase in Ca2+ -independent NOS activity was observed in WEM from EAE 111 rats compared to those from control animais in the presence of ali the cofactors. Ca2+ -independent NOS activity in WEM homogenates from EAE III rats was not affected by the omission of any of the added cofactors, while the one from control animais was significantly inhibited by the absence of either FAD or BH4 in the incubation media. RT-PCR analysis revealed no significant changes in either nNOS or eNOS mRNA among the groups of rats; however, increased levels of iNOS mRNA were detected in WEM homogenates from EAE 111 in comparison with those from control animais. Western blotting of NT-containing proteins showed two major bands (MW: 53 and 28 kDa) of significantly increased intensity at both stages 11 and 111 in comparison with controls. Serum NO3- levels were found significantly higher in EAE 111 rats than in control animais. The addition of either WEM homogenates or sera from control animais to a NOS preparation led to an exacerbated enzyme inhibition than that observed with WEM homogenates or sera obtained from EAE 111 rats. However, differences between groups disappear after heat-denaturation of both the WEM homogenates and sera. Based on these results we conclude that parallel to a decrease of constitutive Ca2+dependent NOS activity, up-regulation of iNOS occurs during advanced EAE stages, which could be responsible for the observed increase in systemic NO production and the appearance of NT-modified proteins at the CNS leveI. On the other hand, the decreased sensitivity of iNOS to both FAD and BH4, and the absence of a thermolabile circulating NOS inhibitor observed in animais with EAE reflect a complex modulation of NOS activity at different regulatory site levels in this animal model of multiple sclerosis

ASSUNTO(S)

encefalomielite oxido nitrico esclerose multipla inflamação

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