AJP - Cell Physiology, Vol 263, Issue 1 C47-C54, Copyright © 1992 by American Physiological Society
Ammoniagenesis in LLC-PK1 cultures: role of transamination
G. Gstraunthaler, F. Landauer and W. Pfaller
Institute of Physiology, University of Innsbruck, Austria.
The LLC-PK1 renal epithelial cell line has been used as a model system to
study renal ammoniagenesis and its regulation by metabolic acidosis in
vitro. Experiments were performed on confluent LLC-PK1 epithelia grown for
10-14 days in conventional monolayer technique. After the medium pH was
changed from 7.6 to 7.0 for 24-72 h by lowering the bicarbonate
concentration in culture medium, LLC-PK1 cells responded with an adaptive
increase in glutamine consumption and ammonia production. The rates of
glutamine uptake and ammonia generation displayed a ratio of 1:1, i.e., 1
mol ammonia was produced per mole of glutamine consumed. Glutamine
consumption and ammonia formation were paralleled by an equimolar
production of L-alanine, indicating that transamination appears to be the
main ammoniagenic pathway in LLC-PK1 cells. Analysis of the key enzymes of
renal ammoniagenesis, phosphate-dependent glutaminase (PDG) and glutamate
dehydrogenase (GDH), revealed no changes in enzyme activities up to 72 h of
adaptation. Alanine aminotransferase (ALT) activity in LLC-PK1 cells also
remained unchanged during the adaptation period. Because transamination
seems to play a crucial role in channeling the metabolic flux in LLC-PK1
ammoniagenesis, experiments were performed in which transamination was
inhibited by (aminooxy)acetate (AOA). After incubation of control and pH
7.0-adapted LLC-PK1 cultures for 24-72 h in 0.2 mM AOA, no alanine
production was found, but 2 mol of ammonia were formed per mole of
glutamine consumed, again, without adaptive changes in PDG and GDH
activities.(ABSTRACT TRUNCATED AT 250 WORDS)
