Biochemical and Ultrastructural Studies of Cultured Rat Astroglial Cells

Sensenbrenner, M.; Devilliers, Ginette; Bock, Elisabeth; Porte, A

doi:10.1111/j.1432-0436.1980.tb01081.x

Cited by 159 publications

(33 citation statements)

References 24 publications

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“…The presence of GAP-43 immunoreactivity in astrocytes was confirmed with another affinity-purified antibody (see above) with results similar to the ones presented here. The staining was independent of the two methods used to prepare astrocytes for culturing (8,9). No cells in these cultures were stained with anti-NF68 antibody as early as 48 hr after seeding, suggesting that neurons were absent from the cultures.…”

Section: Resultsmentioning

confidence: 95%

The 43-kDa neuronal growth-associated protein (GAP-43) is present in plasma membranes of rat astrocytes.

Vitkoviç

Steisslinger

Aloyo

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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One of the neuronal growth-associated proteins, GAP-43 (molecular mass, -43 kDa; pI 4.3), is abundant in growth-cone membranes and corresponds to a major protein kinase C substrate, the 46-kDa phosphoproteiw (pp46), of a growth-cone-enriched subcellular fraction. This protein has the following additional designations (depending on context): B-50 (phospholipid metabolism), F1 (synaptic plasticity), and p57 (calmodulin binding). We show that a protein with the same molecular mass and isoelectric point as GAP-43, which interacts with anti-GAP-43 antibodies on immunoblots, is present in the plasma membranes of cultured neonatal rat cortical astrocytes. Double-immunofluorescence labeling of cells with a serum against glial fibrillary acidic protein and anti-GAP-43 antibody was observed. Furthermore, astrocytic protein was phosphorylated in vitro by protein kinase C and comigrated in two-dimensional PAGE with GAP-43. The data indicate that GAP-43, heretofore believed to be neuronspecific, is present in at least one class of glial cells.Astrocytes constitute about one-half of all glial cells in the central nervous system. Although their precise functions are unknown, data indicate that they may be involved in a symbiotic relationship with neurons and thereby play important roles in neuronal development and function. This suggests that a significant aspect of astrocytic activity involves its plasma membrane. However, astrocytic plasma membranes remain to a large extent biochemically uncharacterized and, as such, their capacity for interacting with their environment is unknown. In this report, the proteins of the astrocytic plasma membrane are characterized by twodimensional PAGE, immunoblotting, and comparison with membranes from other cell types.We have described two brain-specific plasma membrane proteins that are very abundant in rat cortex (1). The smaller polypeptide was identified as growth-associated protein 43 (GAP-43) because of its relative molecular mass (=43 kDa), pI value (pI 4.3), in vitro phosphorylation by protein kinase C (PKC), and reactivity with anti-GAP-43 antibody. A polypeptide with these characteristics has been identified by several groups and designated F1, B-50, and a 46-kDa phosphoprotein (pp46) (2). Since this protein is developmentally regulated and its synthesis increases in regenerating axons, it was named growth-associated protein 43 (3). We show here that a protein with a molecular mass and a pI value similar to those of neuronal GAP-43 is present in the plasma membranes of cultured rat astrocytes. Two anti-GAP-43 antibodies interacted with this protein on immunoblots and mediated fluorescent labeling of cultured astrocytes. It is currently believed that GAP-43 is exclusively a neuronal protein. The presence of GAP-43 in astrocytic membranes may have important implications for the role of this protein in nervous system development.

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Section: Resultsmentioning

confidence: 95%

The 43-kDa neuronal growth-associated protein (GAP-43) is present in plasma membranes of rat astrocytes.

Vitkoviç

Steisslinger

Aloyo

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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“…Vimentin, GFA protein and actin are principal components of non-ionic-detergent-resistant cytoskeleton preparations from astrocytes analyzed by polyacrylamide gel electrophoresis [4,5]. When the differentiation of astrocytes in culture is enhanced with Bt,cAMP, the concentration of G F A protein markedly increases in parallel with an increase in the number of glial filaments [6].…”

Section: Proteolytic Degradation Of Vimentin and Glial Fibrillary Acimentioning

confidence: 99%

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

Ciesielski-Treska

Goetschy

Aunis

1984

European Journal of Biochemistry

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Vimentin and glial fibrillary acidic (GFA) protein in rat astrocytes in primary culture were identified with their respective specific antisera on electroblots of one-dimensional and two-dimensional polyacrylamide gels. Each antiserum revealed respectively vimentin (58 kDa, p15.3), GFA protein (50 kDa, PI 6-5.8) and several more acidic, lower molecular mass vimentin-immunoreactive and GFA protein-immunoreactive polypeptides. These were more apparent when astrocyte proteins were extracted in the absence of EGTA and in the presence of Ca2+ ions suggesting that the lower immunoreactive polypeptides result from a Ca2 +-sensitive proteolytic degradation of vimentin and GFA protein. The extent of proteolysis of GFA protein was higher when the differentiation of astrocytes was induced with 1,N6-dibutyryl-adenosine 3',5'-monophosphate (Bt,cAMP). In contrast, the proteolysis of vimentin was not affected by the state of differentiation of astrocytes. The results indicate the existence of at least two distinct degradative pathways for intermediate filament proteins in astrocytes in primary culture and suggest that in differentiated astrocytes the activity of proteinase(s) involved in the degradation of GFA protein increases.

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“…a peak of EBEE activity, mostly contained in PZ and P3 subcellular fractions; (ii) the 19th DIC primary culture conditioned medium markedly enhanced EBEE activity of the cell lines. It is noteworthy that the concentration of db-cAMP we used was similar to that utilized by other workers who investigated the effect of this compound on 'in vitro cellular models' [26-281, and in particular cultured glial cells [14,29].…”

Section: Discussionmentioning

confidence: 99%

“…Astrocyte primary cultures derived from neonatal rat brain hemispheres were obtained according to Sensenbrenner et al [14]. The cells were seeded at a density of 4 x lo6 cells on 80 mm diameter culture dishes (Falcon 3003, USA).…”

Section: Primary Cultures Of Astrocytesmentioning

confidence: 99%

Ethanolamine base exchange enzymatic activities in spontaneous transformed glial cell lines Effect of dibutyryl cyclic AMP treatment

1988

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Cultured astrocytes derived from neonatal rats (normal cells) displayed maximal ethanolamine base exchange enzymatic activity (EBEE) when cultures reached confluency and cells almost ceased to divide. At this stage, ethanolamine phosphotransferase (EPT) and choline base exchange enzyme (CBEE) activities reached a plateau. In spontaneously transformed glial cells, no differential activity variation either between EPT and CBEE, or between EPT and EBEE was observed. The EBEE activity was mainly localized in the microsomal fraction and was completely absent from plasma membranes. Dibutyryl cyclic AMP (db-CAMP) treatment of the transformed cells reversed the pattern of these activities to that of normal cells. Moreover, treatment of the transformed cells with medium conditioned by normal astroblasts markedly increased EBEE activity. This study demonstrates that (i) variation of EBEE activity during cell growth differs in normal and in transformed cultured glial cells.(ii) EBEE activity may bc modulated via both db-CAMP and normal cell conditioned medium. Our findings suggest a possible implication of EBEE in the maturation and contact inhibition of cell growth.

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Biochemical and Ultrastructural Studies of Cultured Rat Astroglial Cells

Cited by 159 publications

References 24 publications

The 43-kDa neuronal growth-associated protein (GAP-43) is present in plasma membranes of rat astrocytes.

The 43-kDa neuronal growth-associated protein (GAP-43) is present in plasma membranes of rat astrocytes.

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

Ethanolamine base exchange enzymatic activities in spontaneous transformed glial cell lines Effect of dibutyryl cyclic AMP treatment

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