Temporal profiles of the in vitro phosphorylation rate and immunocontent of glial fibrillary acidic protein (GFAP) after kainic acid-induced lesions in area CA1 of the rat hippocampus: demonstration of a novel phosphoprotein associated with gliosis.
The in vitro phosphorylation rate and immunocontent of glial fibrillary acidic protein was studied in slices of area CA1 of the rat hippocampus after stereotaxic injection of 1 nmol of kainic acid. For controls the contralateral hippocampus was injected with saline. Hippocampal tissue was incubated with [32P]phosphate and analysed by two-dimensional electrophoresis for phosphorylation rate and by immunoblotting for immunocontent. Both these parameters decreased during the first 4 days after injection and then started to increase at 10 days and continued to increase until at least 84 days. Except for a small excess of phosphorylation rate at 28 days, the relationship between immunocontent and in vitro phosphorylation rate of glial fibrillary acidic protein remained constant, indicating that the reactive gliosis was not associated with hypo- or a major hyperphosphorylation of this protein. Histology showed a pronounced loss of CA1 pyramidal cells 1 day after injection. At 28 days after injection the pyramidal cells had disappeared and only a few abnormal neurones were present. In contrast, immunocytochemistry after 28 days showed a marked increase in astrocytes reacting positive to the antibody in the strata radiatum and lacunosum moleculare. Besides glial fibrillary acidic protein the expression of several other proteins was upregulated as a result of the injection of kainic acid. These included phosphovimentin and an unknown phosphoprotein designated pp25 which co-migrated on 2-D gels with a prominent phosphoprotein expressed in primary cultures of astrocytes. Pp25 was expressed in lesioned tissue more frequently than phosphovimentin and with a time course that started earlier. Of particular interest was the expression of pp25 in the contralateral saline-injected hippocampus 1 day after injection of kainic acid. It is possible that pp25 will prove to be a sensitive marker of gliosis.
Lenz
G
G
Departamento de Bioquímica, Instituto de Ciências Básicas de Saúde,Porto Alegre, RS, Brazil.
Manozzo
L
L
Gottardo
S
S
Achaval
M
M
Salbego
C
C
Rodnight
R
R
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity*; D005904:Glial Fibrillary Acidic Protein / Q000276:immunology / Q000378:metabolism*; D005911:Gliosis / Q000139:chemically induced / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D007608:Kainic Acid / Q000633:toxicity*; D007700:Kinetics; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D015854:Up-Regulation / Q000187:drug effects; D014746:Vimentin / Q000378:metabolism
D018690:Excitatory Amino Acid Agonists; D005904:Glial Fibrillary Acidic Protein; D010750:Phosphoproteins; D014746:Vimentin; D007608:Kainic Acid