title: Ammoniagenesis in renal cell culture. Lack of extracellular ammoniagenesis at the apical surface of LLC-PK1 epithelia.
abstract: In renal ammoniagenesis, two major pathways of glutamine metabolism have been described: (i) intracellular metabolism by phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase and (ii) extracellular metabolism by phosphate-independent glutaminase. The latter has been identified as the hydrolytic activity of the apically membrane-bound gamma-glutamyl transpeptidase (gamma-GT). The growth properties of cultured renal epithelia enable the study of in vitro extracellular metabolic properties occurring at the apical epithelial surface in the culture dish. Therefore, confluent epithelia of the LLC-PK1 renal epithelial cell line were used to elucidate the role of extracellular (apical) hydrolysis of glutamine by gamma-GT in LLC-PK1 ammonia production. To distinguish between intra- and extracellular metabolism of glutamine, confluent LLC-PK1 epithelia were incubated with either D-glutamine as substrate, which cannot be metabolized intracellularly by PDG, or with L-glutamine and hippurate to stimulate, and AT-125 (acivicin) to inhibit gamma-GT activity, respectively. In addition, cellular uptake of the glutamate, extracellularly formed by gamma-GT, was inhibited by D-aspartate. D-Glutamine (2 mM) did not increase ammonia formation above endogenous production levels, indicating the negligible role of extracellular hydrolysis of glutamine by gamma-GT. After modulating gamma-GT activity by hippurate or AT-125, almost identical ammonia production rates were found within the various experimental protocols, further confirming that extracellular metabolism of glutamine does not significantly contribute to LLC-PK1 ammoniagenesis.
authors.affiliation: Institute of Physiology, University of Innsbruck, Austria.
authors.forename: G
authors.initials: G
authors.lastname: Gstraunthaler
authors.forename: F
authors.initials: F
authors.lastname: Landauer
authors.forename: W
authors.initials: W
authors.lastname: Pfaller
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism*; D002460:Cell Line; D003470:Culture Media; D005971:Glutamates / Q000493:pharmacokinetics; D018698:Glutamic Acid; D005973:Glutamine / Q000493:pharmacokinetics; D006626:Hippurates / Q000494:pharmacology; D007555:Isoxazoles / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000596:Amino Acids; D003470:Culture Media; D005971:Glutamates; D006626:Hippurates; D007555:Isoxazoles; D005973:Glutamine; D018698:Glutamic Acid; D000641:Ammonia; D005723:gamma-Glutamyltransferase; C006303:acivicin; C030514:hippuric acid