Ammoniagenesis in LLC-PK1 cultures: role of transamination.
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)
Gstraunthaler
G
G
Institute of Physiology, University of Innsbruck, Austria.
Landauer
F
F
Pfaller
W
W
D000138:Acidosis / Q000378:metabolism; D000625:Aminooxyacetic Acid / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism*; D002460:Cell Line; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D000637:Transaminases / Q000378:metabolism*
D005973:Glutamine; D000625:Aminooxyacetic Acid; D000641:Ammonia; D000637:Transaminases