Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes

CO 2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14 CO 2 and the incorporation of 14 C into medium or cell extract products was determined. After chromatographic separation of 14 C‐labelled products, the fractions of 14 C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 m M increased pyruvate carboxylase flux as observed by increases in the 14 C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 m M almost doubled CO 2 fixation. When 25 m M bicarbonate plus 0.5 m M pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 m M bicarbonate plus 5 m M pyruvate, the rate of appearance of [ 14 C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α‐ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 m M ) had no effect on net 14 CO 2 fixation, but did alter the distribution of 14 C‐labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14 C incorporation). Low rates of conversion of α‐[ 14 C]ketoglutarate to [ 14 C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α‐ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α‐ketoglutarate to glutamate may frequently be the rate‐limiting step in this process of glutamate synthesis.