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Research ArticleArticle

Regulation of the Glial Na+-Dependent Glutamate Transporters by Cyclic AMP Analogs and Neurons

Brian D. Schlag, Joanna R. Vondrasek, Muhammad Munir, Avtandil Kalandadze, Olga A. Zelenaia, Jeffrey D. Rothstein and Michael B. Robinson
Molecular Pharmacology March 1998, 53 (3) 355-369; DOI: https://doi.org/10.1124/mol.53.3.355
Brian D. Schlag
Children’s Seashore House, Children’s Hospital of Philadelphia, Departments of Pediatrics and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, 1 2
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Joanna R. Vondrasek
Children’s Seashore House, Children’s Hospital of Philadelphia, Departments of Pediatrics and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, 1 2
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Muhammad Munir
Children’s Seashore House, Children’s Hospital of Philadelphia, Departments of Pediatrics and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, 1 2
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Avtandil Kalandadze
Children’s Seashore House, Children’s Hospital of Philadelphia, Departments of Pediatrics and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, 1 2
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Olga A. Zelenaia
Children’s Seashore House, Children’s Hospital of Philadelphia, Departments of Pediatrics and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, 1 2
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Jeffrey D. Rothstein
Children’s Seashore House, Children’s Hospital of Philadelphia, Departments of Pediatrics and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, 1 2
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Michael B. Robinson
Children’s Seashore House, Children’s Hospital of Philadelphia, Departments of Pediatrics and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, 1 2
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Abstract

Sodium-dependent transport into astrocytes is critical for maintaining the extracellular concentrations of glutamate below toxic levels in the central nervous system. In this study, the expression of the glial glutamate transporters GLT-1 and GLAST was studied in primary cultures derived from cortical tissue. In primary astrocytes, GLAST protein levels were approximately one half of those observed in cortical tissue, but GLT-1 protein was present at very low levels compared with cortical tissue. Maintenance of these astrocytes in medium supplemented with dibutyryl-cAMP (dbcAMP) caused a dramatic change in cell morphology, increased GLT-1 and GLAST mRNA levels ≈5-fold, increased GLAST protein ≈2-fold, and increased GLT-1 protein ≥8–20-fold. These increases in protein expression were accompanied by 2-fold increases in the V max andK m values for Na+-dependentl-[3H]glutamate transport activity. Although GLT-1 is sensitive to inhibition by dihydrokainate in heterologous expression systems, no dihydrokainate sensitivity was observed in astrocyte cultures that expressed GLT-1. Biotinylation with a membrane-impermeant reagent, separation of the biotinylated/cell surface proteins, and subsequent Western blotting demonstrated that both GLT-1 and GLAST were present at the cell surface. Coculturing of astrocytes with neurons also induced expression of GLT-1, which colocalized with the glial specific marker, glial fibrillary acidic protein. Neurons induced a small increase in GLAST protein. Several studies were performed to examine the mechanism by which neurons regulate expression of the glial transporters. Three different protein kinase A (PKA) antagonists did not block the effect of neurons on glial expression of GLT-1 protein, but the addition of dbcAMP to mixed cultures of neurons and astrocytes did not cause GLT-1 protein to increase further. This suggests that neurons do not regulate GLT-1 by activation of PKA but that neurons and dbcAMP regulate GLT-1 protein through convergent pathways. As was observed with GLT-1, the increases in GLAST protein observed in cocultures were not blocked by PKA antagonists, but unlike GLT-1, the addition of dbcAMP to mixed cultures of neurons and astrocytes caused GLAST protein to increase ≈2-fold. Neurons separated from astrocytes with a semipermeable membrane increased GLT-1 protein, indicating that the effect of neurons was mediated by a diffusible molecule. Treatment of cocultures with high concentrations of eitherN-methyl-d-aspartate or glutamate killed the neurons, caused GLT-1 protein to decrease, and caused GLAST protein to increase. These studies suggest that GLT-1 and GLAST protein are regulated independently in astrocyte cultures and that a diffusible molecule secreted by neurons induces expression of GLT-1 in astrocytes.

Footnotes

    • Received September 11, 1997.
    • Accepted November 14, 1997.
  • Send reprint requests to: Dr. Michael Robinson, 502N Abramson Pediatric Research Building, 34th & Civic Center Blvd., Philadelphia, PA 19104-4318. E-mail:robinson{at}pharm.med.upenn.edu

  • This work was supported by Grants NS29868 and HD26979 (M.B.R), NS33958 (J.D.R.), and NS36465 (M.B.R., J.D.R.).

  • 1 B.D.S. and J.R.V. contributed equally to this study.

  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 53 (3)
Molecular Pharmacology
Vol. 53, Issue 3
1 Mar 1998
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Research ArticleArticle

Regulation of the Glial Na+-Dependent Glutamate Transporters by Cyclic AMP Analogs and Neurons

Brian D. Schlag, Joanna R. Vondrasek, Muhammad Munir, Avtandil Kalandadze, Olga A. Zelenaia, Jeffrey D. Rothstein and Michael B. Robinson
Molecular Pharmacology March 1, 1998, 53 (3) 355-369; DOI: https://doi.org/10.1124/mol.53.3.355

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Research ArticleArticle

Regulation of the Glial Na+-Dependent Glutamate Transporters by Cyclic AMP Analogs and Neurons

Brian D. Schlag, Joanna R. Vondrasek, Muhammad Munir, Avtandil Kalandadze, Olga A. Zelenaia, Jeffrey D. Rothstein and Michael B. Robinson
Molecular Pharmacology March 1, 1998, 53 (3) 355-369; DOI: https://doi.org/10.1124/mol.53.3.355
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