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.

In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca2+ and Na+ into the neuron. To maintain Na+ homeostasis, the excess Na+ entering through the ion channel should be removed by Na+,K+‐ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na+,K+‐ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate‐induced activation of Na+,K+‐ATPase was dose dependent: It was appreciable (37%) at 0.1 µM and peaked (85%) at 100 µM. The increase in Na+,K+‐ATPase activity by glutamate was prevented by MK‐801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12‐myristate 13‐acetate, an activator of protein kinase C, indicating that activation of Na+,K+‐ATPase is due to decreased phosphorylation by protein kinase C. W‐7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na+,K+‐ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na+,K+‐ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na+,K+‐ATPase.

Acute ammonia toxicity is mediated by activation of NMDA receptors and is prevented by chronic moderate hyperammonaemia. The aim of this work was to assess whether the protective effect of chronic hyperammonaemia is due to impaired activation of the NMDA receptor. It is shown that chronic hyperammonaemia in rats decreases the binding of [3H]MK-801 to synaptosomal membranes from the hippocampus but not the amount of NMDAR1 receptor protein as determined by immunoblotting. In primary cultures of cerebellar neurons, long-term treatment with 1 mM ammonia also decreased significantly the binding of [3H]MK-801. These results suggest that ammonia impairs NMDA receptor activation. To confirm this possibility we tested the effect of long-term treatment of the cultured neurons with 1 mM ammonia on three well known events evoked by activation of the NMDA receptor: neuronal death induced by glutamate, increase in aspartate aminotransferase activity and increase in free intracellular [Ca2+]. Long-term treatment with ammonia prevented noticeably the effects of glutamate or NMDA on all these parameters. These results indicate that long-term treatment of neurons with 1 mM ammonia leads to impaired function of the NMDA receptor, which cannot be activated by glutamate or NMDA. Activation of protein kinase C by a phorbol ester restored the ability of the NMDA receptor to be activated in neurons treated with ammonia. This suggests that ammonia impairs NMDA receptor function by decreasing protein kinase C-dependent phosphorylation.