Role of specific aminotransferases in de novo glutamate synthesis and redox shuttling in the retina

In this study aminotransferase inhibitors were used to determine the relati ve importance of different aminotransferases in providing nitrogen for de n ovo glutamate synthesis in the retina. Aminooxyacetate, which inhibits all aminotransferases, blocked de novo glutamate synthesis from (HCO3)-C-14 - b y more than 60%. Inhibition of neuronal cytosolic branched chain amino acid transamination by gabapentin or branched chain amino acid transport by the L-system substrate analog, 2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic ac id, lowered total de novo synthesis of glutamate by 30%, suggesting that br anched chain amino acids may account for half of the glutamate nitrogen con tributed by transamination reactions. L-cycloserine, an inhibitor of alanin e aminotransferase, inhibited glutamate synthesis less than 15% when added in the presence of 5 mM pyruvate but 47% in the presence of 0.2 mM pyruvate . Although high levels of pyruvate blunted the inhibitory effectiveness of L-cycloserine, the results indicate that, under physiological conditions, a lanine as well as branched chain amino acids are probably the predominant s ources of glutamate nitrogen in ex vivo retinas. The L-cycloserine results were also used to evaluate activity of the malate/aspartate shuttle. In thi s shuttle, cytosolic aspartate (synthesized in mitochondria) generates cyto solic oxaloacetate that oxidizes cytosolic NADH via malate dehydrogenase. B ecause L-cycloserine inhibits cytosolic but not mitochondrial aspartate ami notransferase, L-cycloserine should prevent the utilization of aspartate bu t not its generation, thereby increasing levels of C-14- aspartate. Instead , L-cycloserine caused a significant decline in C-14-aspartate. The results suggest the possibility that shuttle activity is low in retinal Muller cel ls. Low malate/aspartate shuttle activity may be the molecular basis for th e high rate of aerobic glycolysis in retinal Muller cells. (C) 2001 Wiley-L iss, Inc.