Regulation of protein phosphorylation in astrocyte cultures by external calcium ions: specific effects on the phosphorylation of glial fibrillary acidic protein (GFAP), vimentin and heat shock protein 27 (HSP27)

The effect of external Ca2+ ([Ca2+](e)) on the incorporation of [P-32] into total protein, cytoskeletal proteins and the heat shock protein HSP27, was studied in primary cultures of astrocytes from the rat hippocampus. Zero [Ca2+](e) increased total P-32-incorporation into astrocyte protein and when this was normalized to 100%, incorporation was significantly increased into glial fibrillary acidic protein (GFAP), vimentin (VIM) and HSP27. The difference in total P-32-incorporation between zero [Ca2+](e) and 1 mM [Ca2+](e) was reversed by incubation of the cells with the protein phosphatase inhibitor okadaic acid in the range 1-10 nM; higher concentrations of okadaic acid (50-100 nM) further increased total P-32-incorporation. In zero [Ca2+](e) the non-specific channel blocker Co2+ (1 mM) decreased total P-32-incorporation by approximately 30%. The results were compared with a previous study [S.T. Wofchuk, R. Rodnight, Age-dependent changes in the regulation by external calcium ions of the phosphorylation of glial fibrillary acidic protein in slices of rat hippocampus, Dev. Brain Res. 85 (1995) 181-186] in which it was shown that in immature hippocampal slices zero [Ca2+](e) compared with 1 mM [Ca2+](e) increased P-32-incorporation into GFAP without changing total incorporation. The difference between the results for total P-32-incorporation obtained in cultured astrocytes and immature brain tissue was found to be related to the concentration of [Ca2+](e) in the medium since in slices concentrations of [Ca2+](e) higher than 1 mM progressively decreased total incorporation. The difference may reflect a higher Ca2+-permeability of the plasma membrane in cultured astrocytes and/or to the complex structure of the slice tissue. In two-dimensional electrophoresis HSP27, in contrast to GFAP and VIM, was separated into 3 immunodetectable isoforms only two of which were normally phosphorylated. After labelling in the presence of okadaic acid both immunodetectable and phosphorylated HSP27 focussed as a single polypeptide. Phorbol dibutyrate (1 mu M) and zero [Ca2+](e) stimulated the phosphorylation of both isoforms, but in the case of zero [Ca2+](e) the effect on the more acidic isoform was proportionally greater. (C) 1999 Published by Elsevier Science B.V. All rights reserved.