A C-13-NMR STUDY ON THE INFLUXES INTO THE TRICARBOXYLIC-ACID CYCLE OF A RENAL EPITHELIAL-CELL LINE, LLC-PK1/CL4 - THE METABOLISM OF [2-C-13]GLYCINE, L-[3-C-13]ALANINE AND L-[3-C-13]ASPARTIC ACID IN RENAL EPITHELIAL-CELLS

Perchloric acid extracts of LLC-PK1/Cl4 cells, a renal epithelial cell line, incubated with either [2-13C]glycine L-[3-13C]alanine, or D,L-[3-13C]aspartic acid were investigated by 13C-NMR spectroscopy. All amino acids, except labelled glycine, gave rise to glycolytic products and tricarboxylic acid cycle (TCA) intermediates. For the first time, we also observed activity of .gamma.-glutamyltransferase activity and glutathione synthetase activity in LLC-PK1 in cells, as is evident from enrichment of reduced glutathione. Time courses showed that only 6% of the labelled glycine was utilized in 30 min, whereas 31% of L-alanine and 60% of L-aspartic acid was utilized during the same period. 13C-NMR was also shown to be a useful tool for the determination of amino acid uptake in LLC-PK1 cells. These uptake experiments indicated that glycine, alanine and aspartic acid are transported into Cl4 cells via a sodium-dependent process. From the relative enrichment of the glutamate carbons, we calculated the activity of pyruvate dehydrogenase to be about 61% when labelled L-alanine was the only carbon source for LLC-PK1/Cl4 cells. Experiments with labelled D,L-aspartic, however, showed that about 40% of C-3 enriched oxaloacetate (arising from a de-amination of aspartic acid) reached the pyruvate pool.