Metabolic Fate of 14C‐Labeled Glutamate in Astrocytes in Primary Cultures

Abstract

The metabolic fate of L-[U-¹⁴C]- and L-[l-¹⁴C]glutamate was studied in primary cultures of mouse astrocytes. Conversion of the uniformly labeled compound to glutamine and aspartate was followed by determination of specific activities after dansylation with [³H]dansyl chloride and subsequent thin layer chromatography of the dansylated amino acids. Metabolic fluxes were calculated from the alterations of specific activities and the pool sizes, which were likewise measured by a dansylation method. Formation of ¹³CO₂ from [l-¹⁴C]glutamate was determined by the trapping of CO₂ in hyamine hydroxide in a gas-tight chamber, which is, in the known absence of glutamate decarboxylase activity in the cultured astrocytes, an unequivocal expression of the metabolic flux via α-ketoglutarate to CO₂ and succinyl-CoA. The metabolic fluxes determined by these procedures amounted to 2.4 nmol/midmg protein for glutamine synthesis, 1.1 nmolimidmg protein for aspartate production, and 4.1 nmollmidmg protein for formation and subsequent decarboxylation of α- ketoglutarate. The latter process was unaffected by virtually complete inhibition of glutamate-oxaloacetic transaminase with aminooxyacetic acid, indicating that the formation of α-ketoglutarate occurs as an oxidative deamination rather than as a transamination. This suggests that the formation of α-ketoglutarate from glutamate represents a net degradation, not an isotopic exchange.