RESUMO
In this study we investigated the effects of alpha-ketoisovaleric (KIV) and alpha-keto-beta-methylvaleric acids (KMV), metabolites accumulating in the inherited neurometabolic disorder maple syrup urine disease (MSUD), on the in vitro incorporation of 32P into intermediate filament (IF) proteins from cerebral cortex of young rats during development (9-21 days of age) We observed that KMV significantly increased the in vitro incorporation of 32P into the IF proteins studied in cortical slices of 12-day-old rats through the PKA and PKCaMII, with no alteration at the other ages. In contrast, KIV was ineffective in altering the phosphorylating system associated with IF proteins at all ages examined. A similar effect on IF phosphorylation was achieved by incubating cortical slices with gamma-aminobutiric acid (GABA). Furthermore, by using specific GABA antagonists, we verified that KMV induced a stimulatory effect on IF phosphorylation of tissue slices from 12-day-old rats mediated by GABA(A) and GABA(B) receptors. In conclusion, our results indicate the involvement of the GABAergic system in the alterations of IF phosphorylation caused by KMV, one of the branched-chain keto acids accumulating in MSUD.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Filamentos Intermediários/metabolismo , Cetoácidos/farmacologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Benzilaminas/farmacologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Eletroforese em Gel de Poliacrilamida/métodos , Inibidores Enzimáticos/farmacologia , Antagonistas GABAérgicos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Hemiterpenos , Técnicas In Vitro , Isoquinolinas/farmacologia , Isótopos de Fósforo/metabolismo , Fosforilação/efeitos dos fármacos , Radioquímica/métodos , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Vimentina/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Procedures for the preparation of high- and low-salt Triton insoluble cytoskeletal fractions from rat brain suitable for studying in vitro phosphorylation by endogenous kinases and phosphatases are described. The high-salt Triton insoluble cytoskeletal fraction is enriched in neurofilament subunits (NF-H, NF-M and NF-L), vimentin and glial fibrillary acidic protein (GFAP), while the low-salt Triton insoluble cytoskeletal fraction contains detergent insoluble cytoskeletal elements such as intermediate filament subunits and tubulins. One of our approaches is to incubate cerebral cortex slices with [32P]orthophosphate before the cytoskeletal fraction extraction, which allows the in vitro phosphorylation of cytoskeletal constituents in an intact intracellular environment. On the other hand, we also incubate low- or high-salt cytoskeletal fractions previously prepared with [gamma(32)P]ATP. By doing so, we are able to study the direct effects of substances on the kinase and phosphatase activities associated with the cytoskeletal fraction. Moreover by using specific activators or inhibitors of protein kinases and phosphatases we can obtain more detailed information on the alterations provoked by these substances. These approaches are useful for the investigation of the neurotoxic effects of various drugs and metabolites affecting the cytoskeletal-associated phosphorylation system in the brain.
Assuntos
Córtex Cerebral/metabolismo , Proteínas do Citoesqueleto/isolamento & purificação , Histocitoquímica/métodos , Técnicas de Cultura de Órgãos/métodos , Frações Subcelulares/química , Trifosfato de Adenosina , Animais , Córtex Cerebral/citologia , Proteínas do Citoesqueleto/química , Inibidores Enzimáticos/farmacologia , Feminino , Proteína Glial Fibrilar Ácida/química , Proteína Glial Fibrilar Ácida/isolamento & purificação , Histocitoquímica/instrumentação , Masculino , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/isolamento & purificação , Fosfatos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Isótopos de Fósforo , Fosforilação , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Ratos , Ratos Wistar , Vimentina/química , Vimentina/isolamento & purificaçãoRESUMO
In this study we investigated the effects of alpha-ketoisocaproic acid (KIC), the main keto acid accumulating in the inherited neurometabolic disorder maple syrup urine disease (MSUD), on the in vitro incorporation of 32P into intermediate filament (IF) proteins from cerebral cortex of rats during development. KIC decreased the in vitro incorporation of 32P into the IF proteins studied up to day 12, had no effect on day 15, and increased this phosphorylation in cortical slices of 17- and 21-day-old rats. A similar effect on IF phosphorylation was achieved along development by incubating cortical slices with glutamate. Furthermore, the altered phosphorylation caused by the presence of KIC in the incubation medium was mediated by the ionotropic receptors NMDA, AMPA and kainate up to day 12 and by NMDA and AMPA in tissue slices from 17- and 21-day-old rats. The results suggest that alterations of IF phosphorylation may be associated with the neuropathology of MSUD.