title: Acute Hyperammonemia Induces NMDA-Mediated Hypophosphorylation of Intermediate Filaments Through PP1 and PP2B in Cerebral Cortex of Young Rats.
abstract: In the present work, we studied the effects of toxic ammonia levels on the cytoskeleton of neural cells, with emphasis in the homeostasis of the phosphorylating system associated with the intermediate filaments (IFs). We used in vivo and in vitro models of acute hyperammonemia in 10- and 21-day-old rats. In the in vivo model, animals were intraperitoneally injected with ammonium acetate (7 mmol/Kg), and the phosphorylation level of the cytoskeletal proteins was analyzed in the cerebral cortex and hippocampus 30 and 60 min after injection. The injected ammonia altered the IF phosphorylation of astrocytes (GFAP and vimentin) and neurons (neurofilament subunits of low, middle, and high molecular weight, respectively: NFL, NFM, and NFH) from cerebral cortex of 21-day-old rats. This was a transitory effect observed 30 min after injection, recovering 30 min afterward. Phosphorylation was not altered in the cerebral cortex of 10-day-old pups. The homeostasis of hippocampal IFs was preserved at the studied ages and times. In the in vitro model, cortical slices of 10- and 21-day-old rats were incubated with 0.5, 1, or 5 mM NH4Cl, and the phosphorylation level of the IF proteins was analyzed after 30 min. The IF phosphorylation was not altered in cortical slices of 10-day-old rats; however, in cortical slices of 21-day-old pups, 5 mM NH4Cl induced hypophosphorylation of GFAP and vimentin, preserving neurofilament phosphorylation levels. Hypophosphorylation was mediated by the protein phosphatases 1 (PP1) and 2B (PP2B), and this event was associated with Ca(2+) influx via N-methyl-D-aspartate (NMDA) glutamate receptors. The aim of this study is to show that acute ammonia toxicity targets the phosphorylating system of IFs in the cerebral cortex of rats in a developmentally regulated manner, and NMDA-mediated Ca(2+) signaling plays a central role in this mechanism. We propose that the disruption of cytoskeletal homeostasis could be an endpoint of the acute hyperammonemia in the developing brain. We believe that these results contribute for better understanding the molecular basis of the ammonia toxicity in brain.
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, Porto Alegre, RS, CEP 90035-003, Brazil.
authors.forename: Rônan Vivian
authors.initials: RV
authors.lastname: Carvalho
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, Porto Alegre, RS, CEP 90035-003, Brazil.
authors.forename: Fernanda
authors.initials: F
authors.lastname: da Silva Ferreira
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, Porto Alegre, RS, CEP 90035-003, Brazil.
authors.forename: Luana
authors.initials: L
authors.lastname: Heimfarth
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, Porto Alegre, RS, CEP 90035-003, Brazil.
authors.forename: Paula
authors.initials: P
authors.lastname: Pierozan
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, Porto Alegre, RS, CEP 90035-003, Brazil.
authors.forename: Carolina
authors.initials: C
authors.lastname: Fernandes
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, Porto Alegre, RS, CEP 90035-003, Brazil. rpureur@ufrgs.br.
authors.forename: Regina
authors.initials: R
authors.lastname: Pessoa-Pureur
mesh_terms: D000085:Acetates; D000375:Aging / Q000378:metabolism; D000643:Ammonium Chloride; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002540:Cerebral Cortex / Q000378:metabolism*; D004195:Disease Models, Animal; D006624:Hippocampus / Q000378:metabolism; D022124:Hyperammonemia / Q000378:metabolism*; D007382:Intermediate Filaments / Q000378:metabolism*; D016202:N-Methylaspartate / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D017208:Rats, Wistar; D013997:Time Factors; D046509:Tissue Culture Techniques
chemical_list: D000085:Acetates; D000643:Ammonium Chloride; D016202:N-Methylaspartate; D019703:Calcineurin; D054645:Protein Phosphatase 1; C018824:ammonium acetate; D002118:Calcium
keywords: Calcium; Glutamate receptors; Hyperammonemia; Hypophosphorylation; Intermediate filaments; Protein phosphatases