Pathways and regulation of ammoniagenesis by the LLC-PK1 cells in culture.
LLC-PK1 kidney epithelial cells grown under the condition of continuous rocking exhibit a variety of differentiated functions of proximal tubular epithelium, including pH-modulated ammoniagenesis. To further determine their value as a model system, we investigated the pathways of ammoniagenesis under both normal conditions and acid-base manipulations. Pulse-chase studies with carbon 14-labeled glutamine demonstrated a marked delay in glutamine conversion to glutamate, indicating that glutamine deamidation is a critical rate-limiting step, and also provided evidence for metabolism of the glutamine carbon skeleton by the tricarboxylic acid cycle. Ammonia and alanine were the predominant nitrogen metabolites of glutamine at all pH conditions, and the stoichiometry suggested that glutamate is metabolized through both glutamate dehydrogenase and glutamate transaminase at pH 7.4. Increased ammonia production in response to a low pH was associated with increased flux through phosphate-dependent glutaminase and the glutamate transamination pathway and was accompanied by a fall in intracellular glutamate and alpha-ketoglutarate concentrations, which was similar to events in the intact kidney. Studies with the inhibitors acivicin and amino oxyacetate suggested that the gamma-glutamyltranspeptidase and glutamine transamination pathways are inconsequential in LLC-PK1 cells. The phosphate-dependent glutaminase pathway appears to play a predominant role in the regulation of ammoniagenesis. The similarity in ammonia metabolism with other in vitro and in vivo models suggests that LLC-PK1 cells will be a useful system for investigating renal ammoniagenesis and the intracellular signals that modulate this process.
Sahai
A
A
Department of Internal Medicine, University of Michigan, Ann Arbor.
Cole
L A
LA
Tannen
R L
RL
D000409:Alanine / Q000096:biosynthesis; D000625:Aminooxyacetic Acid / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D002460:Cell Line; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D005973:Glutamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007555:Isoxazoles / Q000494:pharmacology; D007656:Ketoglutaric Acids / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological
D000963:Antimetabolites; D005971:Glutamates; D007555:Isoxazoles; D007656:Ketoglutaric Acids; D005973:Glutamine; D000625:Aminooxyacetic Acid; D018698:Glutamic Acid; D000641:Ammonia; C006303:acivicin; D000409:Alanine