Nitrogen shuttling between neurons and glial cells during glutamate synthesis

The relationship between neuronal glutamate turnover, the glutamate/glutami ne cycle and de novo glutamate synthesis was examined using two different m odel systems, freshly dissected rat retinas ex vivo and in vivo perfused ra t brains. In the ex vivo rat retina, dual kinetic control of de novo glutam ate synthesis by pyruvate carboxylation and transamination of alpha -ketogl utarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide t he alpha -amino group to glutamate. Measurements of synthesis of C-14-gluta mate and of C-14-glutamine from (HCO3)-C-14 have shown that C-14-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mM. The s pecific radioactivity of C-14-glutamine indicated that similar to 30% of gl utamine was derived from (CO2)-C-14 fixation. Using gabapentin, an inhibito r of the cytosolic branched-chain aminotransferase, synthesis of 14C-glutam ate and C-14-glutamine from (HCO3-)-C-14 was inhibited by 31%. These result s suggest that transamination of alpha -ketoglutarate to glutamate in Mulle r cells is slow, the supply of branched-chain amino acids may limit flux, a nd that branched-chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysi s suggests that the glutamate/glutamine cycle accounts for 15% of total neu ronal 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 (HCO3-)-C-14. C-14-metabolite s in brain extracts were measured to determine net incorporation of (CO2)-C -14 and specific radioactivity of glutamate and glutamine. The results indi cate that 23% of glutamine in the brain in vivo is derived from (CO2)-C-14 fixation. Using published values for whole brain neuronal glutamate turnove r, we calculated that the glutamate/glutamine cycle accounts for similar to 60% of total neuronal turnover. Finally, differences between glutamine/glu tamate cycle rates in these two model systems suggest that the cycle is clo sely linked to neuronal activity.