Santiago Grisolia

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.

A sensitive and convenient micromethod for estimation of urea, citrulline, and carbamyl derivatives

Abstract A simple, rapid, reproducible method for the millimicromolar to micromolar estimation of urea, citrulline, carbamyl aspartate, and related compounds is presented. The method obeys Beers law.

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.

Inhibitors of protein kinase C prevent the toxicity of glutamate in primary neuronal cultures

Glutamate-induced neurotoxicity has been proposed to depend on a sustained increase of intracellular free Ca2+ levels. However, the molecular mechanism(s) involved are not well understood. Some results suggest that activation of protein kinase C by the increased levels of Ca2+ could play a role in the mediation of glutamate neurotoxicity. To assess this hypothesis we have tested if the 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H7) and calphostin C, inhibitors of protein kinase C, are able to protect neurons in primary culture from glutamate-induced cell death. It is shown that both H7 and calphostin C prevent nearly completely the death of neurons from cerebellum, even when 2 mM glutamate was used. HA-1004, an inhibitor of cyclic nucleotide-dependent protein kinases, did not protect neurons. The protective effect was maximum at approximately 10 microM H7 and at approximately 10 nM calphostin C. The results reported support the hypothesis that protein kinase C plays a key role in the mediation of glutamate neurotoxicity.

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.

Protective effect of L-carnitine on hyperammonemia

Inborn errors of the urea cycle, liver malfunction and drug‐induced hepatotoxicity are causes of life‐threatening encephalopathies arising from hyperammonemia. L‐Carnitine prevented entirely ammonia toxicity in mice when injected intraperitoneally 30 min before a lethal dose of ammonium acetate. Survival depends on the dose of L‐carnitine injected, e.g., 0, 60, 70, 80 and 100% with 0, 1, 2, 8 and 16 mmol L‐carnitine/kg, respectively. At the highest doses L‐carnitine abolishes the convulsions that accompany acute ammonia intoxication. At lower doses it delayed their onset. The protective effect was associated with a marked decrease of blood ammonia, while in unprotected mice ammonemia was lethal in less than 15 min. When sustained hyperammonemia was induced by urease injections, protection was also obtained. The mechanism of protection is under investigation, however, since L‐carnitine facilitates fatty acid entry into mitochondria, possibly ATP or reducing equivalents are increased.

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.

Changes in glutathione in acute and chronic alcohol intoxication.

After 5 hours of acute ethanol intoxication, the levels of reduced glutathione of liver, heart, kidney and brain decreased to 60, 71, 70 and 83% of controls, respectively. The decrease was less when animals were pretreated with pyrazole. Pretreatment with disulfiram potentiated the effect of ethanol. Levels of GSSG were not altered by these treatments. Chronic ethanol administration, in a liquid diet, also produced a decrease in GSH levels. The lowest levels were observed at 4 weeks in liver (65%). However, there was a slow recovery to 75% and 80%, respectively, at 10 and 14 weeks of treatment. Withdrawal from alcohol administration after 10 and 14 weeks intoxication resulted in a complete recovery in GSH levels within 24 hours. Similar changes were seen in other tissues, although they were less marked than in liver, and no changes were observed in GSSG. To clarify the changes in glutathione levels, we measured the main enzymes connected with the synthesis (γ-glutamylcysteine synthetase + glutathione synthetase) and utilization (γ-glutamyl transpeptidase) of glutathione. In liver, the activity of the glutathione synthesizing system was unchanged after 4 weeks, but increased to 140% within 10 weeks; γ-glutamyl transpeptidase was slightly elevated (120%) at 4,10 and 14 weeks. Similar changes in these enzymes were observed in kidney and brain. γ-glutamyl transpeptidase was elevated in serum (120%) at 4 weeks, but reached 140% within 10 weeks. The levels of the glutathione synthesizing system returned to normal in all tissues within 2 days after alcohol withdrawal; however, γ-glutamyl transpeptidase levels did not revert to normal until one week after withdrawal.