María-Dolores Miñana

Acute ammonia toxicity is mediated by the NMDA type of glutamate receptors

Previous experiments in our laboratory suggested that ammonium toxicity could be mediated by the NMDA type of glutamate receptors. To assess this hypothesis we tested if MK‐801, a specific antagonist of the NMDA receptor, is able to prevent ammonium toxicity. Mice and rats were injected i.p. with 12 and 7 mmol/kg of ammonium acetate, respectively, 73% of the mice and 70% of the rats died. However, when the animals were injected i.p. with 2 mg/kg of MK‐801, 15 min before ammonium injection, only 5% of the mice and 15% of the rats died. The remarkable protection afforded by MK‐801 indicates that ammonia toxicity is mediated by the NMDA receptor.

Brain ATP Depletion Induced by Acute Ammonia Intoxication in Rats Is Mediated by Activation of the NMDA Receptor and Na+, K+‐ATPase

Abstract: 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 ammonium‐induced 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 ammonia‐induced 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 ammonia‐induced 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.

NMDA Receptor Antagonists Prevent Acute Ammonia Toxicity in Mice

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.

Nicotine prevents glutamate-induced proteolysis of the microtubule-associated protein MAP-2 and glutamate neurotoxicity in primary cultures of cerebellar neurons.

The aim of this work was to assess whether nicotine prevents glutamate neurotoxicity in primary cultures of cerebellar neurons, to try to identify the receptor mediating the protective effect and to shed light on the step of the neurotoxic process which is prevented by nicotine. It is shown that nicotine prevents glutamate and NMDA neurotoxicity in primary cultures of cerebellar neurons. The protective effect of nicotine is not prevented by atropine, mecamylamine or dihydro-beta-erythroidine, but is slightly prevented by hexamethonium and completely prevented by tubocurarine and alpha-bungarotoxin, indicating that the protective effect is mediated by activation of alpha7 neuronal nicotinic receptors. Moreover, alpha-bungarotoxin potentiates glutamate neurotoxicity, suggesting a tonic prevention of glutamate neurotoxicity by basal activation of nicotinic receptors. Nicotine did not prevent glutamate-induced rise of free intracellular calcium nor depletion of ATP. Nicotine prevents glutamate-induced proteolysis of the microtubule-associated protein MAP-2 and disaggregation of the neuronal microtubular network. The possible mechanism responsible for this prevention is discussed.

Glutamate Induces a Calcineurin-Mediated Dephosphorylation of Na+,K+-ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract: 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.

Nitroarginine, an Inhibitor of Nitric Oxide Synthetase, Attenuates Ammonia Toxicity and Ammonia-Induced Alterations in Brain Metabolism

We have proposed that acute ammonia toxicity is mediated by activation of the N-methyl-D-aspartate type of glutamate receptors. MK-801, a selective antagonist of these receptors, prevents death of animals induced by acute ammonia intoxication as well as ammonia-induced depletion of ATP. It seems therefore that, following activation of the N-methyl-D-aspartate receptors, the subsequent events in ammonia toxicity should be similar to those involved in glutamate neurotoxicity. As it has been shown that inhibitors of nitric oxide synthetase such as nitroargnine prevent glutamate toxicity, we have tested whether nitroarginine prevents ammonia toxicity and ammonia-induced alterations in brain energy and ammonia metabolites. It is shown that nitroarginine prevents partially (≈50%), but significantly death of mice induced by acute ammonia intoxication. Nitroarginine also prevents partially ammonia-induced depletion of brain ATP. It also prevents completely the rise in glucose and pyruvate and partially that in lactate. Injection of nitroarginine alone, in the absence of ammonia, induces a remarkable accumulation of glutamine and a decrease in glutamate. The results reported indicate that nitroarginine attenuates acute ammonia toxicity and ammonia-induced alterations in brain energy metabolites. The effects of MK-801 and of nitroarginine are different, suggesting that ammonia can induce nitric oxide synthetase by mechanisms other than activation of N-methyl-D-aspartate receptors.

Ammonia Prevents Activation of NMDA Receptors by Glutamate in Rat Cerebellar Neuronal Cultures

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.

Induction of Rat Brain Tubulin Following Ammonium Ingestion

Abstract: The effect of oral administration of ammonium acetate for 2, 15, 30, and 100 days on protein synthesis in rat brain was investigated. Although protein synthesis changes were modest, i.e., maximal increase of 24%, there was induction of synthesis and accumulation of a protein with an Mr of 55,000. We show, on the basis of its position on two‐dimensional electrophoresis and its immunological reactivity, that this protein is tubulin. Its content increased by 33% as determined by isolation of tubulin after 15 days of oral administration of ammonium and to 49% after 100 days as determined by quantitative immunoblotting.

Ammonium Injection Induces an N-Methyl-d-Aspartate Receptor-Mediated Proteolysis of the Microtubule-Associated Protein MAP-2

Abstract: We have shown previously that chronic hyperammonemia increases, in brain, the polymerization of microtubules that is regulated mainly by the level and state of phosphorylation of microtubule‐associated protein 2 (MAP‐2). Activation of the N‐methyl‐d‐aspartate (NMDA) receptor dephosphorylates MAP‐2. Because we have found that acute ammonia toxicity is mediated by the NMDA receptor, we have tested the effect of high ammonia levels on MAP‐2 in brain. Microtubules isolated from rats injected intraperitoneally with 6 mmol/kg ammonium acetate showed a marked decrease of MAP‐2. Also, the amount of MAP‐2 in brain homogenates, determined by immunoblotting. was markedly reduced, presumably by proteolysis. The content of MAP‐2 was decreased by ∼75% 1‐2 h after ammonium injection and returned to normal values after 4 h. Proteolysis of MAP‐2 was prevented completely by injection of 2 mg/kg MK‐801, a specific antagonist of the NMDA receptor, suggesting that proteolysis is mediated by activation of this receptor. l‐Carnitine, which protects rats against ammonia toxicity, also prevented MAP‐2 degradation. Because activation of the NMDA receptor increases [Ca2+]i, we determined whether rat brain contains a Ca2+‐dependent protease that selectively degrades MAP‐2. We show that there is a cytosolic Ca2+‐dependent protease that degrades MAP‐2, but no other brain proteins. The protease has been identified tentatively as calpain I, for it is inhibited by a specific inhibitor of this protease. Our results suggest that ammonium injection activates the NMDA receptor, leading to an increase in [Ca2+]i, which activates calpain I. This, in turn, selectively degrades MAP‐2. Possible implications in chronic hyperammonemic states and in the mechanism of ammonia toxicity are discussed.

Chronic hyperammonemia prevents changes in brain energy and ammonia metabolites induced by acute ammonium intoxication

Acute ammonia toxicity has been attributed to the depletion of energy metabolite intermediates. Ingestion of an ammonium containing diet produces hyperammonemia and protects rats against acute ammonium intoxication. We have tested the effect of chronic hyperammonemia on the brain contents of energy and ammonia metabolite intermediates and on the effect on these contents of acute ammonia intoxication (i.p. injection of 7 mmol/kg of ammonium acetate). Chronic hyperammonemia was induced in rats by feeding them a diet containing 20% ammonium acetate. Control rat were fed the same diet without addition of ammonium acetate. It is shown that chronic hyperammonemia did not affect the content of most metabolites, the only remarkable changes are the increases of the contents of ammonia (46%), glutamine (81%), acetoacetate (31%) and of the mitochondrial NAD+/NADH ratio (32%) as well as the marked decrease of beta-hydroxybutyrate (by 86%). Chronic hyperammonemia prevents most changes in metabolites induced by acute ammonium intoxication (i.p. injection of 7 mmol/kg of ammonium acetate). In control rats it was a marked breakdown of glycogen and increased contents of glucose, lactate and pyruvate, with decreased cytosolic NAD+/NADH ratio and beta-hydroxybutyrate and ATP contents. These changes were nearly completely prevented in hyperammonemic rats. In controls, ammonia increased 12.8-fold while glutamate and aspartate decreased by approximately 40% and glutamine and alanine raised by 37% and 93%, respectively; in hyperammonemic rats ammonia increased 6.9-fold while glutamate, glutamine and alanine were not significantly affected. Also the mitochondrial NAD+/NADH ratio raised by 18-fold in controls and by 6-fold in hyperammonemic rats. These results indicate that chronic hyperammonemia markedly prevents the alterations of the contents of energy and ammonia metabolites induced by acute ammonium intoxication.