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Vol. 53, Issue 3, 355-369, March 1998
Children's Seashore House, Children's Hospital of Philadelphia,
Departments of Pediatrics and Pharmacology, University of Pennsylvania,
Philadelphia, Pennsylvania,
19104-4318 (B.D.S., J.R.V., M.M., A.K.,
O.A.Z., M.B.R.), and
Department of Neurology, The Johns Hopkins
University, Baltimore, Maryland 21287 (J.D.R)
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 Vmax and Km values for
Na+-dependent
L-[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 either
N-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.
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