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.

Modulation of glutamine synthesis in cultured astrocytes by nitric oxide.

1. Previous results suggest that glutamine synthesis in brain could be modulated by nitric oxide. The aim of this work was to assess this possibility.

Activation of the metabotropic glutamate receptor mGluR5 prevents glutamate toxicity in primary cultures of cerebellar neurons.

1-Aminocyclopentane-trans-1,3-dicarboxylic acid, an agonist of the metabotropic glutamate receptors 1, 2, 3 and 5, prevents neurotoxicity of glutamate and of N-methyl-D-aspartate in primary cultures of cerebellar neurons. The aim of this work was to assess which of the metabotropic glutamate receptors (mGluRs) is responsible for the protective effect. We tested the protective effects of selective agonists for each type of receptor.

Determination of intracellular ATP in primary cultures of neurons.

The aim of this work was to develop and characterize a quick and simple procedure to determine the intracellular content of ATP in monolayer primary cultures of neurons. The baseline was to use the minimum amount of cells which still provides reproducible results. The first step consists of releasing intracellular ATP from the cells.

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.

Carnitine and choline derivatives containing a trimethylamine group prevent ammonia toxicity in mice and glutamate toxicity in primary cultures of neurons.

Carnitine prevents acute ammonia toxicity in animals. We propose that acute ammonia toxicity is mediated by activation of N-methyl-D-aspartate receptors and have shown that carnitine prevents glutamate neurotoxicity. The aim of this work was to assess whether other compounds containing a trimethylamine group are able to prevent ammonia toxicity in mice and/or glutamate toxicity in primary neuronal cultures.

H7, an inhibitor of protein kinase C, prevents serum-induced phosphorylation of Raf and MAP kinase in neuroblastoma cells.

We have previously shown that 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), an inhibitor of protein kinase C, inhibits proliferation of neuroblastoma cells in culture. We have now tested whether the effect of H7 is mediated by MAP kinase and Raf. It is shown that, in Neuro 2a cells, activation of protein kinase C by addition of 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (PMA), leads to phosphorylation of Raf and Mitogen-activated protein kinase (MAP kinase).

Protein kinase C isoforms and cell proliferation in neuroblastoma cells.

The expression of protein kinase C isoforms in the neuroblastoma cell line Neuro 2a has been studied. It is shown that Neuro 2a cells express alpha, delta, epsilon and zeta PKCs. Inhibition of cell proliferation by using protein kinase C inhibitors (H7 or calphostin C) or medium without glutamine affects markedly the pattern of PKC isoforms.

Effects of acute hyperammonemia in vivo on oxidative metabolism in nonsynaptic rat brain mitochondria.

The effects of hyperammonemia induced in vivo by injecting rats with ammonium acetate on oxidative phosphorylation, malate-aspartate shuttle, some related enzyme activities and metabolite levels in brain mitochondria were studied ex vivo. Rats were found to be either ammonia-sensitive (showing convulsions) or ammonia-resistant (without convulsions) after intraperitoneal injection of ammonium acetate (7 mmol/kg). Ammonium acetate administration to ammonia-sensitive rats led to inhibition of State 3 rates of brain mitochondria utilizing pyruvate, glutamate, isocitrate, and succinate as substrates and to decreased respiratory control index.

Glutamate induces a calcineurin-mediated dephosphorylation of Na+,K(+)-ATPase that results in its activation in cerebellar neurons in culture.

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.

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.

Lack of correlation between glutamate-induced depletion of ATP and neuronal death in primary cultures of cerebellum.

The aim of this work was to identify, using primary cultures of cerebellar neurons, the receptors involved in glutamate-induced depletion of ATP and to assess whether there is a correlation between glutamate-induced ATP depletion and neuronal death. Glutamate induced a rapid depletion of ATP (40% decrease at 5 min). After 60 min incubation with 1 M glutamate ATP content decreased by 60-70%.

Prenatal exposure of rats to ammonia impairs NMDA receptor function and affords delayed protection against ammonia toxicity and glutamate neurotoxicity.

The aim of this work was to assess whether perinatal hyperammonemia impairs the function of NMDA receptors and whether this impairment affords protection against acute ammonia toxicity and glutamate and NMDA neurotoxicity. Rats were exposed to ammonia during the prenatal and lactation periods by feeding the female rats an ammonium-containing diet since day 1 of pregnancy. After weaning (at postnatal day 21), the pups were fed a normal diet with no ammonia added.

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.

Protein kinase C inhibitors, H7 and calphostin C, inhibit induction of DNA synthesis by cytosolic extracts of exponentially growing neuroblastoma cells in isolated nuclei.

Cytoplasmic extracts from proliferating Neuro-2a cells contain a protein factor, ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. Cytoplasmic extracts derived from quiescent-made Neuro-2a cells contain none or very little ADR activity, but this activity can be generated after a brief exposure of cytosolic extracts to a membrane-enriched fraction derived from exponentially growing Neuro-2a cells. ADR activity appears at the beginning of the S phase of the cell cycle.

Brain ATP depletion induced by acute ammonia intoxication in rats is mediated by activation of the NMDA receptor and Na+,K(+)-ATPase.

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.

High ammonia levels decrease brain acetylcholinesterase activity both in vivo and in vitro.

We have tested the effect of ammonium injection on the activity of acetylcholinesterase in rat brain. Fifteen minutes after ip injection of 7 mmol/kg of ammonium acetate, the activity of acetylcholinesterase in brain was reduced significantly. The inhibitory effect varied in a wide range, with a maximum decrease of 60%, and was proportional to the concentration of ammonia reached in the brain.

L-carnitine increases the affinity of glutamate for quisqualate receptors and prevents glutamate neurotoxicity.

We have shown that acute ammonia toxicity is mediated by activation of the NMDA type of glutamate receptors. Although it is well known that L-carnitine prevents acute ammonia toxicity, the underlying molecular mechanism is not clear. We suspected that L-carnitine would prevent ammonia toxicity by preventing the toxic effects of glutamate.