High extracellular glutamate modulates expression of glutamate transporters and glutamine synthetase in cultured astrocytes

Abstract

Astroglial cells clear extracellular glutamate through the glutamate transporters, GLT-1 and GLAST, and subsequently convert the incorporated glutamate into glutamine by the enzyme, glutamine synthetase (GS). Several forms of acute brain injury are associated with the increased expression of GS and the decreased expression of GLT-1 and/or GLAST, eventually leading to the accumulation of excitotoxic extracellular glutamate concentrations. Although of clinical interest, the actual trigger of these injury-related changes of glial glutamate turnover remains unknown. Our present studies provide evidence that increases in extracellular glutamate, as present in many brain injuries, are sufficient to modulate the expression of glutamate transporters and GS. Subjecting cultured cortical astrocytes to glutamate concentrations of 0.5-20 mM resulted in a 25% loss of GLT-1 and GLAST protein levels after 24 h; GLT-1 and GLAST levels maximally decreased by 40% and 75%, respectively, after 72 h. This decline was not due to astroglial cell death, since glutamate up to 50 mM did not affect the survival of cultured astrocytes within 72 h. Major astrocytic cell death, however, occurred in cultures maintained under severe (4% O(2)), but not mild (9% O(2)), hypoxia, as well as in the presence of aspartate (>or=20 mM). Glutamate at >or=1 mM induced a prolonged increase of GS expression in contrast to glutamate transporters. Neither the decline of glutamate transporter expression nor the increase in GS expression induced by high extracellular glutamate was further modulated by mild hypoxia. Whereas the stimulatory influences of glutamate on GS expression were prevented by the non-competitive NMDA receptor antagonist, MK801, the inhibitory influences on glutamate transporter expression were neither sensitive to MK801, the non-competitive mGluR5 antagonist, MTEP, nor the non-competitive AMPA receptor antagonist, GYKI52466, implying that glutamate controls glial glutamate transport by a glutamate receptor-independent mechanism.