Ammoniagenesis by cultured human renal cortical epithelial cells: study with 15N.
The metabolic fate of 15N-labeled glutamine and glutamate in cultured human renal cortical epithelial cells was investigated. The main goal was to elucidate the major pathways of ammoniagenesis depending on varying H+ concentration. Incubations at pH 7.4 or 6.8 were conducted with either 1 mM [5-15N]glutamine, [2-15N]glutamine, [15N]glutamate, or L-[2-15N]-gamma-glutamylmethylamide. The results demonstrate that acute acidosis had little effect on total ammonia generation from glutamine. However, 15NH3 formation from [5-15N]glutamine was significantly higher at pH 7.4 compared with pH 6.8. Conversely, at pH 6.8, 15NH3 production from either [2-15N]-glutamine or [15N]glutamate was twofold higher than at pH 7.4. Thus the observations indicate that acute acidosis had little effect on net ammonia production from glutamine due to decreased flux through glutaminase and concomitant increased flux through glutamate dehydrogenase. When L-[2-15N]-gamma-glutamylmethylamide was utilized as the sole substrate, significantly higher amounts of 15NH3 and 15N-labeled amino acids were formed at pH 6.8 compared with pH 7.4. Addition of either 1 mM pyruvate or alpha-ketoglutarate significantly decreased 15NH3 and increased 15N-amino acid formation from either [2-15N]glutamine or [2-15N]-gamma-glutamylmethylamide. The metabolism of either substrate via transamination reaction was significantly stimulated at acidic pH, presumably due to a depleted pool of alpha-ketoglutarate during the course of the incubations. The data indicate that in addition to glutaminase I and glutamate dehydrogenase, the glutamine aminotransferase (glutaminase II) pathway exists in cultured human renal cells. The data suggest that glutamate dehydrogenase flux and/or the alpha-ketoglutarate dehydrogenase reaction may have an important regulatory role in ammoniagenesis from glutamine and/or glutamate in human kidney during acute acidosis.
Nissim
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I
Division of Biochemical Development and Molecular Dieases, Children's Hospital of Philadelphia, Pennsylvania.
States
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D000641:Ammonia / Q000096:biosynthesis*; D002478:Cells, Cultured; D004848:Epithelium / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000166:cytology*; D009587:Nitrogen Isotopes; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid
D005971:Glutamates; D009587:Nitrogen Isotopes; D011773:Pyruvates; D005973:Glutamine; C014574:gamma-glutamylmethylamide; D018698:Glutamic Acid; D000641:Ammonia; D019289:Pyruvic Acid