Mechanisms of ammonia-induced cell death in rat cortical neurons: roles of NMDA receptors and glutathione.
The occurrence, nature and prevention of ammonia-induced cell death were assayed in cultured primary cortical neurons from newborn rats. Treatment with 1-10 mM ammonium chloride for 24 or 48 h, dose-dependently decreased neuronal survival (MTT assay) and GSH/GSSG ratio in the cultures, whereas total GSH content was significantly reduced only with 10mM ammonia. Treatment with a glutathione synthesis inhibitor, buthionyl sulfoximine (BSO) (10 microM), decreased the GSH content and GSH/GSSG ratio to a degree similar to that of 10 mM ammonia, but it did not decrease cell survival in control cells. This indicates that glutathione depletion per se is not a cause of ammonia-induced neuronal death. However, ammonia-induced decrease of cell viability was attenuated by incubation with glutathione diethyl ester (GEE), which transiently increased the intracellular GSH level in both control and ammonia-treated cells. Neuronal survival in the presence of ammonia was partly improved by the NMDA receptor antagonists MK-801 and APV. Morphological analysis revealed that ammonia treatment causes both apoptotic and non-apoptotic neuronal death, the former not being inhibited by MK-801. Apoptosis was the dominant type of cell death at 10mM ammonia, as concluded both from morphologic examination and the absence of survival improvement in the presence of GABA+nipecotic acid or taurine, model anti-excitotoxic treatments of cortical neurons. The mechanism underlying apoptosis may include inhibition of a survival kinase, Akt, whose activatory phosphorylation at Ser473 is reduced in neurons treated with 10 mM, but not 1 mM ammonia.
Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Pasteura St. 3, 02-093 Warsaw, Poland.
Agata
A
Klejman
Michał
M
Wegrzynowicz
Erzsebet M
EM
Szatmari
Barbara
B
Mioduszewska
Michał
M
Hetman
Jan
J
Albrecht
D000641:Ammonia / Q000378:metabolism / Q000633:toxicity*; D000643:Ammonium Chloride / Q000633:toxicity; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism* / Q000503:physiopathology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D006501:Hepatic Encephalopathy / Q000378:metabolism / Q000503:physiopathology; D022124:Hyperammonemia / Q000378:metabolism / Q000503:physiopathology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000378:metabolism*
D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D009498:Neurotoxins; D011518:Proto-Oncogene Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D000643:Ammonium Chloride; D000641:Ammonia; C083627:glutathione diethyl ester; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D005978:Glutathione