Cultures of rat astrocytes challenged with a steady supply of glutamate: new model to study flux distribution in the glutamate-glutamine cycle.
Glutamate metabolism in astrocytes was studied using an experimental setup that simulates the role of neurons (glutamate producers and glutamine consumers) by the addition of glutaminase to the culture medium. Thereby, a steady supply of glutamate was imposed at the expense of glutamine, and the stress intensity was manipulated by changing the glutaminase concentration. Glutamate supply rates in the range 8-23 nmol/min/mg protein were examined for periods of up to 48 h. When the glutamate supply rate exceeded the uptake rate of this amino acid, a transient increase in the extracellular concentration of glutamate was observed. In response to this stress, the fluxes through the glutamate transporter and glutamine synthetase were increased considerably, and the extracellular concentration of glutamate was eventually restored to a low level. The increased levels of glutamine synthetase were demonstrated by immunoblotting analysis. The effect on glutamate metabolism of the transaminase inhibitor, aminooxyacetic acid (AOAA), and of NH4Cl was also investigated. The supply of glutamate caused a concomitant reduction in the levels of phosphocreatine, phosphoethanolamine, and phosphocholine without affecting the ATP pool. Glutamine synthetase was shown to be is a key element in the control of glutamate metabolism in astrocytic cultures. The metabolic fate of glutamate depends greatly on the time of endurance to the challenge: in naive cells, glutamate was primarily metabolized through the transaminase pathway, while in well-adapted cells glutamate was converted almost exclusively through glutamine synthetase.
Fonseca
Luís L
LL
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Monteiro
Miguel A R
MA
Alves
Paula M
PM
Carrondo
Manuel J T
MJ
Santos
Helena
H
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002490:Central Nervous System / Q000378:metabolism*; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004983:Ethanolamines / Q000378:metabolism; D045604:Extracellular Fluid / Q000187:drug effects / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D005972:Glutaminase / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010725:Phosphocreatine / Q000378:metabolism; D010767:Phosphorylcholine / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013312:Stress, Physiological / Q000378:metabolism / Q000503:physiopathology; D009435:Synaptic Transmission / Q000502:physiology; D000637:Transaminases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D004791:Enzyme Inhibitors; D004983:Ethanolamines; D010725:Phosphocreatine; D005973:Glutamine; D010767:Phosphorylcholine; D018698:Glutamic Acid; C005448:phosphorylethanolamine; D000637:Transaminases; D005972:Glutaminase; D005974:Glutamate-Ammonia Ligase