Previous experiments in our luboratory suggested that ammonium toxici1y could be medialed by the NMDA 1ype of glutamate receptors. To assess this hypothesis we tcstcd if MK-501. a specific antagonist of the NMDA receptor, is able to prevent ammonium toxicity. Mice and ra1s were injected i.p. with 12 and 7 mmol/kg of ammonium acetalc. rcspL%tively. 73% of the mice and 70% of the ruts died. However. when the animals were injected i.p. with 2 mg/kg of MK-501. I5 min before ammonium injection, only 5% of 1hc mice and 15% of 1hc rats died. The remarkable protection afforded by MK-SOi indicates (ha1 ammonia toxici1y is mediated by the NMDA receptor.Ammonia 1oxicity; NMDA reccplor; MK-901; Hyperammoncmia
Injection of large doses of ammonia into rats leads to depletion of brain ATP. However, the molecular mechanism leading to ATP depletion is not clear. The aim of the present work was to assess whether ammoniuminduced depletion of ATP is mediated by activation of the NMDA receptor . It is shown that injection of MK-801, an antagonist of the NMDA receptor, prevented ammoniainduced ATP depletion but did not prevent changes in glutamine, glutamate, glycogen, glucose, and ketone bodies . Ammonia injection increased Na',K+-ATPase activity by 76%. This increase was also prevented by previous injection of MK-801 . The molecular mechanism leading to activation of the ATPase was further studied. Na+,K+-ATPase activity in samples from ammonia-injected rats was normalized by "in vitro" incubation with phorbol 12-myristate 13-acetate, an activator of protein kinase C. The results obtained suggest that ammoniainduced ATP depletion is mediated by activation of the NMDA receptor, which results in decreased protein kinase C-mediated phosphorylation of Na+,K+-ATPase and, therefore, increased activity of the ATPase and increased consumption of ATP.
We proposed that acute ammonia toxicity is mediated by activation of NMDA receptors. To confirm this hypothesis we have tested whether different NMDA receptor antagonists, acting on different sites of NMDA receptors, prevent death of mice induced by injection of 14 mmol/Kg of ammonium acetate, a dose that induces death of 95% of mice. MK-801, phencyclidine and ketamine, which block the ion channel of NMDA receptors, prevent death of at least 75% of mice. CPP, AP-5, CGS 19755, and CGP 40116, competitive antagonists acting on the binding site for NMDA, also prevent death of at least 75% of mice. Butanol, ethanol and methanol which block NMDA receptors, also prevent death of mice. There is an excellent correlation between the EC50 for preventing ammonia-induced death and the IC50 for inhibiting NMDA-induced currents. Acute ammonia toxicity is not prevented by antagonists of kainate/AMPA receptors, of muscarinic or nicotinic acetylcholine receptors or of GABA receptors. Inhibitors of nitric oxide synthase afford partial protection against ammonia toxicity while inhibitors of calcineurin, of glutamine synthetase or antioxidants did not prevent ammonia-induced death of mice. These results strongly support the idea that acute ammonia toxicity is mediated by activation of NMDA receptors.
Rats were fed the following diets: standard (20% protein), high-protein (80%), protein-free, standard plus ammonium and protein-free plus ammonium for six weeks. The standard plus ammonium diet was prepared to contain ammonia equivalent to that supplied by the high-protein diet. Addition of ammonium acetate (20% by mass) to the 20% protein or protein-free diets results in 2.3-and 10-fold increases of urea excretion respectively, without increase of carbamoyl-phosphate synthase.Supplementation of the standard diet with ammonium increases the mitochondria1 content of acetylglutamate from 830 to 1590 pmol/mg protein, and of the protein-free diet from 130 to 1040 pmol/mg. However, ingestion of ammonium did not increase the activity of acetylglutamate synthase. Therefore the efflux of acetylglutamate from mitochondria was determined. After 30 min at 37°C liver mitochondria from rats on standard diet released 61 YO of the initial acetylglutamate while mitochondria from animals on standard plus ammonium diet released only 20%. These results indicate that ingestion of ammonium increases the content of acetylglutamate in rat liver by decreasing its efflux from mitochondria. This effect is similar to that produced in mice by a high protein diet (Morita et al. (1 982) J. Biochem. (Tokyo) 91, 563 -5691. However while the high-protein diet increases carbamoylphosphate synthase content, the ammonium diet does not.The concentration of ammonia in blood and liver is maintained at nearly constant levels. Although ammonia is a precursor of a number of compounds, it is toxic when in excess. Ureotelic species get rid of ammonia mainly via the urea cycle, which is under complex regulation. Two main mechanisms controlling the urea cycle in response to variations of dietary intake of nitrogen have been described. There is a long-term adaptation in which the hepatic content of the five urea-cycle enzymes responds to the protein intake and is reflected in the rate of urea excretion [I -21. There is also a short-term regulatory mechanism in which acetylglutamate, the physiological allosteric activator of carbamoyl-phosphate synthase I [3 -51, plays a key role. Acetylglutamate levels in liver change rapidly in response to protein ingestion [6], arginine administration [7] or amino acid loads [XI. Variations of acetylglutamate levels correlate well with the urogenic capacity of the animal [6].We have used a high-protein diet (80%) as well as standard (20% protein) or protein-free diets, supplemented or not with ammonium acetate (20% by mass). The effects of such diets on urea excretion, carbamoyl-phosphate synthase activity and liver mi tochondrial acetylglutamate content were determined. The effect of supplementation of standard diet with ammonium on synthesis and degradation of acetylglutamate in liver was also studied. On the basis of the results obtained, we report here the existence of a long-term adaptative mechanismCorrespondence to S. Grisolia in response to ingestion of large amounts of ammonium in which urea production increase...
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