Physiological levels of ammonia regulate glutamine synthesis from extracellular glutamate in astrocyte cultures.
The effect of ammonia on glutamate accumulation and metabolism was examined in astrocyte cultures prepared from neonatal rat cortices. Intact astrocytes were incubated with 70 microM L-[14C(U)]glutamate and varying amounts of ammonium chloride. The media and cells were analyzed separately by HPLC for amino acids and labelled metabolites. Extracellular glutamate was reduced to 8 microM by 60 min. Removal of glutamate from the extracellular space was not altered by addition of ammonia. The rate of glutamine synthesis was increased from 3.6 to 9.3 nmol/mg of protein/min by addition of 100 microM ammonia, and intracellular glutamate was reduced from 262 to 86 nmol/mg of protein after 30 min. The metabolism of accumulated glutamate was matched nearly perfectly by the synthesis of glutamine, and both processes were proportional to the amount of added ammonia. The transamination and deamination products of glutamate were minor metabolites that either decreased or remained unchanged with increasing ammonia. Thus, ammonia addition stimulates the conversion of glutamate to glutamine in intact astrocyte cultures. At physiological concentrations of ammonia, glutamine synthesis appears to be limited by the rate of glutamate accumulation and the activity of competing reactions and not by the activity of glutamine synthetase.
Waniewski
R A
RA
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000096:biosynthesis; D001253:Astrocytes / Q000378:metabolism*; D002478:Cells, Cultured; D005110:Extracellular Space / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D005973:Glutamine / Q000096:biosynthesis*; D007700:Kinetics
D005971:Glutamates; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D000641:Ammonia