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Clearance of synaptic glutamate by glial cells is required for the normal function of excitatory synapses and for prevention of neurotoxicity. Although the regulatory role of glial glutamate transporters in glutamate clearance is well established, little is known about the influence of glial glutamate metabolism on this process. This study examines whether glutamine synthetase (GS), a glial-specific enzyme that amidates glutamate to glutamine, affects the uptake of glutamate. Retinal explants were incubated in the presence of [(14)C]glutamate and glutamate uptake was assessed by measurement of the amount of radioactively labeled molecules within the cells and the amount of [(14)C]glutamine released to the medium. An increase in GS expression in Müller glial cells, caused by induction of the endogenous gene, did not affect the amount of glutamate accumulated within the cells, but led to a dramatic increase in the amount of glutamine released. This increase, which was directly correlated with the level of GS expression, was dependent on the presence of external sodium ions, and could be completely abolished by methionine sulfoximine, a specific inhibitor of GS activity. Our results demonstrate that GS activity significantly influences the uptake of glutamate by the neural retina and suggest that this enzyme may represent an important target for neuroprotective strategies.

Basic fibroblast growth factor (bFGF) was recently shown to promote the survival of neural cells and tissues, raising hopes for its therapeutic potential in degenerative disorders of the CNS. Here we examine the effect of bFGF on the expression of glutamine synthetase, a key enzyme in the detoxification of the neurotransmitter glutamate. Expression of this enzyme is regulated by systemic glucocorticoids and, in chick neural retina tissue, is restricted to Müller glial cells. We report that exogenous supply of bFGF to retinal explants inhibits hormonal induction of glutamine synthetase expression. This inhibition appears to be mediated by the c-Jun protein which accumulated, in response to bFGF, exclusively in Müller glial cells. Ischemic conditions, which reportedly stimulate the release of endogenous bFGF, also led to an increase in c-Jun protein and a decline in glutamine synthetase expression. This decline could be competitively prevented by a soluble fibroblast growth factor receptor but not by a soluble epidermal growth factor receptor. The finding that endogenous release of bFGF or its exogenous supply down-regulates glutamine synthetase expression suggests that in addition to its reported neuroprotective effect, bFGF may exacerbate glutamate mediated neurotoxicity through direct down-regulation of glutamine synthetase.

Inducibility by glucocorticoids of the glutamine synthetase gene in chicken embryo retina and the transcriptional activity of the glucocorticoid receptor (GR) greatly increase between embryonic days 6 and 10 (E6, E10), although the level of GR does not markedly change during that time. This apparent discrepancy was investigated by examining the pattern of GR expression in undifferentiated E6 retina and in E10 retina, which consists mostly of differentiated cells. Two GR isoforms, 90 and 95 kDa, were found to be expressed at both of these ages at a similar total level but in different proportions: in E6 retina the level of the 90-kDa isoform was higher, whereas in E10 retina the 95-kDa receptor was higher. However, following treatment of the retinas with cortisol, the 95-kDa isoform became the predominant receptor at both ages. Immunohistochemical analysis revealed that the cellular localization of GR markedly changed in the course of development: in the undifferentiated E6 retina GR was expressed in virtually all cells, whereas in the more differentiated E10 and E12 retina, GR was detected only in Müller glia cells. The latter represent approximately 20% of the cells in this tissue and are the only cells in which glucocorticoid hormone induces the glutamine synthetase gene. We suggest that the compartmentalization of GR in Müller glia is a major aspect of the mechanism that modulates receptor activity during retina development and results in the temporal increase in the inducibility of glutamine synthetase and its specific localization in Müller glia cells.

Glutamine synthetase (GS) is a differentiation marker of retina glial cell. It is expressed in the chicken neural retina at a particularly high level, is inducible by glucocorticoids and is always confined to Müller glia. This study investigated the molecular basis for tissue and cell-type specific expression of the GS gene. A high level of GS expression in the retina was found to coincide with the accumulation of a relatively high level of GS mRNA in this tissue. The gliatoxic agent alpha-aminoadipic acid, which can selectively destroy glia cells, was used to demonstrate that restriction of GS induction to Müller glia is controlled at a transcriptional level. Cortisol could induce accumulation of GS mRNA and transcription of the GS gene in Müller glia but not in retina neurons. Glia and neurons were also found to differ in their ability to express the glucocorticoid inducible CAT construct, p delta G46TCO, which is controlled by a 'simple GRE' promoter. When introduced into cells of retina tissue, this construct was cortisol-inducible in glia whereas in neurons it was only slightly inducible or not at all. Introduction of a glucocorticoid receptor expression vector into the cells facilitated induction of the CAT construct in neurons. Analysis by immunoblotting revealed that expression of the glucocorticoid receptor protein is predominantly restricted to Müller glia. These results suggest that differential levels of glucocorticoid receptor expression in glia and neurons might be the basis for cell-type specific induction of GS.

Glutamine synthetase is a differentiation marker of the neural retina, whose expression is restricted to Müller glia cells, is inducible by glucocorticoids and is dependent on tissue development. The retina tissue acquires the competence to express GS in response to glucocorticoids with development, although the level of hormone binding activity in the cells does not alter with age. Using CAT constructs that are controlled by "simple GRE" promoters we demonstrated that glucocorticoid receptor transcription activity in retina cells increases with development. The increase in receptor activity correlates directly with the increase in inducibility of the glutamine synthetase gene and inversely with the rate of retina cell proliferation. At early developmental ages, when retina cells are still proliferating, the glucocorticoid receptor is transcriptionally inactive and glutamine synthetase expression cannot be induced. Receptor activity increases progressively with development and by day 12, when cell proliferation ceases, competence for glutamine synthetase induction is high. This competence for glutamine synthetase induction can be repressed by overexpressing the oncogene v-src, which stimulates retina cell proliferation. We discuss possible mechanisms for developmental-dependent modulation of glucocorticoid receptor transcriptional activity.

In chicken embryo retina, competence for induction of the glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] gene by glucocorticoid hormones increases progressively with development; this competence is minimal in 6-day retina (E6) and high by day 10 (E10). Because the level of glucocorticoid receptors (GRs) in the retina does not increase during that time, we investigated whether the transcriptional activity of GR increased between days 6 and 10 of development. The glucocorticoid-inducible chloramphenicol acetyltransferase (CAT) constructs 2GRE-37TK and p delta G46TCO, which contain glucocorticoid-responsive elements attached to a TATA box and to the thymidine kinase promoter, respectively, were transfected into E6 and E10 retinas, and their inducibility was examined. CAT expression could be induced in the transfected E10 retina but was not induced in the transfected E6 retina. However, induction was obtained also in E6 retina after cotransfection with a GR expression vector. Noninducible CAT constructs (pRSV-CAT, pSV2CAT, and pBLCAT2) were expressed at both ages at similar levels. The CAT construct pGS2.1CAT, which is controlled by the upstream sequence of the chicken glutamine synthetase gene, could be induced in E10 retina but was not induced in E6 retina; however, cotransfection with the GR expression vector resulted in induction of pGS2.1CAT also in E6 retina. We interpret these results as showing that the transcriptional activity of GR in embryonic retina is developmentally controlled and suggest that its increase is causally implicated in the development of competence for glutamine synthetase induction.