AJP - Renal Physiology, Vol 253, Issue 6 1243-F1252, Copyright © 1987 by American Physiological Society
Characterization of amino acid metabolism by cultured rat kidney cells: study with 15N
I. Nissim, B. States, M. Yudkoff and S. Segal
Division of Biochemical Development and Molecular Diseases, Children's Hospital of Philadelphia, Pennsylvania.
The present study evaluates the metabolism of glutamine and glutamate by
normal rat kidney (NRK) cells. The major aim was to evaluate the effect of
acute acidosis on the metabolism of amino acid and ammonia formation by
cultured NRK cells. Experiments at either pH 7.0 or 7.4 were conducted with
phosphate-buffered saline supplemented with either 1 mM [5-15N]glutamine,
[2-15N]glutamine, or [15N]glutamate. Incubation with either glutamine or
glutamate as a precursor showed that production of ammonia and glucose was
increased significantly at pH 7.0 vs. 7.4. The disappearance [corrected] of
glutamine and glutamate was linear during a 60-min incubation at either pH.
In experiments with [5-15N]glutamine, we found that approximately 57 and
43% of ammonia N was derived from 5-N of glutamine at pH 7.4 and 7.0,
respectively. Experiments with [2-15N]glutamine or [15N]glutamate indicated
that approximately 43 and 47% of 2-N glutamine and glutamate N utilization,
respectively, was accounted for by ammonia production at pH 7.0. Similarly,
28 and 29% of NH3 was derived from 2-N of glutamine or glutamate N by
activity of glutamate dehydrogenase at pH 7.4. In addition to 15NH3
formation, three major metabolic pathways of [2-15N]glutamine or
[15N]glutamate disposal were identified: 1) transamination reactions
involving the pH-independent formation of [15N] aspartate and [15N]alanine;
2) the synthesis of [6-15NH2]adenine nucleotide, a process more active at
pH 7.4 vs. 7.0; and 3) glutamine synthesis from [15N]glutamate, especially
at pH 7.4. The data indicate that NRK cells in culture consume glutamine
and glutamate and generate ammonia and various amino acids, depending on
the H+ concentration in the media. The studies suggest that these cell
lines may provide a useful model for studying various aspects of the effect
of pH on rat renal ammoniagenesis.
