Nitrogen shuttling between neurons and glial cells during glutamate synthesis

Abstract

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 a-ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the a-amino group to glutamate. Measurements of synthesis of 14 C-glutamate and of 14 C-glutamine from H 14 CO 3 have shown that 14 C-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mM. The speci®c radioactivity of 14 C-glutamine indicated that ,30% of glutamine was derived from 14 CO 2 ®xation. Using gabapentin, an inhibitor of the cytosolic branched-chain aminotransferase, synthesis of 14 C-glutamate and 14 C-glutamine from H 14 CO 3 2 was inhibited by 31%. These results suggest that transamination of a-ketoglutarate to glutamate in Mu È ller cells is slow, the supply of branchedchain amino acids may limit¯ux, 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 H 14 CO 3 2 . 14 C-metabolites in brain extracts were measured to determine net incorporation of 14 CO 2 and speci®c radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from 14 CO 2 ®xation. 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.

Characterization of insulin signaling in rat retina in vivo and ex vivo

“…For ex vivo experiments, rats were anesthetized with pentobarbital sodium at 100 mg/kg and decapitated upon loss of motor reflexes; their retinas were removed by cutting across the cornea, removing the lens, and squeezing the eyeball with forceps to rapidly extract the retina. Retinas were incubated in MEM (Sigma) supplemented with 5 mM pyruvate and 10 mM HEPES for 15 min at 37°C, 5% CO2, and gentle shaking, essentially as described by us and others for the study of retinal metabolism and effects of growth factors (14,33,34). Insulin (10 nM), IGF-I, IGF-II (IGF-binding protein resistant; Upstate Biotechnology, Lake Placid, NY), or vehicle was then added, and these conditions were maintained throughout the duration of the experiment.…”

Diabetic Retinopathy

“…Mü ller cells and astrocytes convey substrates, including lactate and amino acids, from the circulation to neurons and regulate blood-retinal barrier properties (7) and synaptic function (8). Mü ller cells also store glycogen for conversion to lactate, synthesize retinoic acid from retinol, regulate extracellular ion concentrations to modulate plasma membrane polarization/depolarization, participate with neurons in the glutamate/glutamine cycle to control neurotransmission, and protect neurons from glutamate excitotoxicity (9). Glial cells are the interface between the neurons and the vasculature and are thus key regulators of neuronal nutrition and metabolism.…”

The Ammonium-Induced Increase in Rat Brain Lactate Concentration is Rapid and Reversible and is Compatible with Trafficking and Signaling Roles for Ammonium

“…This shuttle requires that fixed nitrogen be returned to the astrocytes. Leucine is rapidly taken up into astrocytes in culture and donates nitrogen for the synthesis of glutamate and glutamine (Yudkoff et al, 1996); it has been suggested that there may be a leucine/a-ketoisocaproic acid shuttle between neurons and astrocytes (Lieth et al, 2001). However, this shuttle would tend to reduce, rather than restore, neuronal glutamate: Yudkoff et al (1996) proposed that it may have a long-term homeostatic role, but it could not contribute to the rapid refilling of synaptic vesicles.…”