The metabolism of glutamic acid by rat cortical astrocyte cultures

The glutamine cycle hypothesis asserts that glutamate released as neurotransmitter is taken up by surrounding glia where it is converted to glutamine. This glutamine then serves to maintain extracellular glutamine which in turn is used to replenish neurotransmitter glutamate pools. The purpose of this dissertation was to examine in detail the regulation of glutamate metabolism in rat cortical astrocyte cultures. Initially, the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS) were evaluated. Rates of {dollar}sp{lcub}14{rcub}{lcub}rm CO{rcub}sb2{dollar} production from (1-{dollar}sp{lcub}14{rcub}{dollar}C) -glutamate and the metabolic fate of ({dollar}sp{lcub}14{rcub}{dollar}C(U)) -glutamate were determined in the presence and absence of the transaminase inhibitor, aminooxyacetic acid, and/or methionine sulfoximine, an inhibitor of GS. Most of the exogenously added glutamate was converted to glutamine and exported into the extracellular medium; however, some glutamate oxidation occurred in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Studies with aminooxyacetic acid showed that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase was the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. The rate of conversion of exogenous glutamate to glutamine was positively correlated with the GS activity of the cultures while the intracellular glutamate levels were inversely related to GS activity. The flux of glutamate through the transamination pathway was highly dependent on the intracellular glutamate concentration. Thus, the GS activity determined not only the rate of glutamine synthesis, but by regulating intracellular glutamate levels, also determined the extent of glutamate oxidation.; Aspects of the regulation and turnover of GS in cultured astrocytes were also examined. Under the conditions examined, the half-life of the enzyme was approximately 48 hours. Dibutyryl cAMP or hydrocortisone caused a 2 to 3-fold increase in steady-state GS activity by increasing the rate of enzyme synthesis, attributable to the associated elevation of GS mRNA levels. The steady-state GS activity of the cultures was inversely related to the concentration of glutamine in the growth medium. The data suggested that high glutamine levels caused an increase in the rate of GS degradation.