Effects of chronic exposure to ammonia on glutamate and glutamine interconversion and compartmentation in homogeneous primary cultures of mouse astrocytes.
Accumulation of radioactivity was studied in primary cultures of mouse astrocytes as a function of time of exposure (4-60 min) to 50 microM glutamate and 200 microM glutamine (initial concentrations), of which either glutamate or glutamine was 14C-labeled. Both the glutamate pool and the glutamine pool were compartmentalized. Initially, by far the major intracellular glutamate pool (> or = 90%) was derived from extracellular glutamate and could be converted to glutamine. This allowed a rather accurate determination of metabolic flux from glutamate to glutamine, which under control conditions amounted to 2.0-2.2 nmol/min per mg protein. After chronic exposure to 3 mM ammonia for 3 days this flux was significantly increased to 3.1-3.6 nmol/min per mg protein. Acute exposure to ammonia caused a smaller, apparent increase, which was not statistically significant. The glutamine content was compartmentalized at all stages of the incubation. It consisted of at least two different pools. One of these was accessible to extracellular glutamine and could be converted to intracellular glutamate (constituting a sizeable fraction of the total glutamate pool after longer incubation), whereas the other constituted endogenously derived glutamine, formed from accumulated glutamate. The specific activity of the precursor pool for glutamate synthesis could not be accurately determined and relatively exact fluxes therefore not be calculated. There was, however, no evidence that chronic exposure to ammonia decreases the rate of glutamine hydrolysis.
Huang
R
R
Department of Pharmacology, University of Saskatchewan, Saskatoon, Canada.
Kala
G
G
Murthy
R K
RK
Hertz
L
L
D000641:Ammonia / Q000633:toxicity*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D005973:Glutamine / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D011865:Radioisotope Dilution Technique; D012044:Regression Analysis; D013997:Time Factors
D002250:Carbon Radioisotopes; D005971:Glutamates; D005973:Glutamine; D018698:Glutamic Acid; D000641:Ammonia