Nitrogen shuttling between neurons and glial cells during glutamate synthesis

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of α-ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the α-amino group to glutamate. Measurements of synthesis of 14C-glutamate and of 14C-glutamine from H14CO3 have shown that 14C-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mm. The specific radioactivity of 14C-glutamine indicated that ∼30% of glutamine was derived from 14CO2 fixation. Using gabapentin, an inhibitor of the cytosolic branched-chain aminotransferase, synthesis of 14C-glutamate and 14C-glutamine from H14CO3− was inhibited by 31%. These results suggest that transamination of α-ketoglutarate to glutamate in Muller cells is slow, the supply of branched-chain amino acids may limit flux, and that branched-chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H14CO3−. 14C-metabolites in brain extracts were measured to determine net incorporation of 14CO2 and specific radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from 14CO2 fixation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for ∼60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.