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Biomedical subjects

V Felipo

Publications and source records attributed to V Felipo.

At least 19 recordsLinked to original sources
PubMed1999

Prevention of glutamate neurotoxicity in cultured neurons by 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6), a scavenger of nitric oxide.

Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive production of nitric oxide (NO). We anticipated that 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6) could act as a scavenger of NO since it contains a position (C-5) highly activated towards nitration reaction. The aim of this work was to assess whether CR-6 acts as an NO scavenger and prevents glutamate neurotoxicity in cultures of cerebellar neurons. It was shown that CR-6 reduced, in a dose-dependent manner, glutamate-induced formation of cGMP (EC50 approximately 15 microM) and prevented glutamate neurotoxicity. The protection was approximately 50% at 3-10 microM and nearly complete at 100 microM. CR-6 did not prevent glutamate-induced activation of NO synthase, but interfered with the glutamate-NO-cGMP pathway at a later step. CR-6 reduced the formation of cGMP induced by S-nitroso-N-acetylpenicillamine (SNAP), an NO-generating agent, indicating that CR-6 acts as a scavenger of NO in cultured neurons. This was further supported by experiments showing that in neurons treated with CR-6 and glutamate, the 5-nitro derivative of CR-6 was formed, as determined by GC-MS analyses. Moreover, in vitro incubation of CR-6 with SNAP also produced the 5-nitroderivative, thus confirming that CR-6 directly reacts with NO. The results reported indicate that CR-6 acts as an NO scavenger in neurons and prevents glutamate neurotoxicity.

Animals↗
PubMed1999

Chronic hyperammonemia in rats impairs activation of soluble guanylate cyclase in neurons and in lymphocytes: a putative peripheral marker for neurological alterations.

Chronic hyperammonemia impairs the glutamate-nitric oxide-cGMP pathway in rat brain in vivo. The aims of this work were to assess whether hyperammonemia impairs modulation of soluble guanylate cyclase, and to look for a peripheral marker for impairment of this pathway in brain. We activated the pathway at different steps using glutamate, SNAP, or YC-1. In control neurons these compounds increased cGMP by 7.4-, 9.7- and 7.2-fold, respectively. In ammonia-treated neurons formation of cGMP induced by glutamate, SNAP, and YC-1 was reduced by 50%, 56%, and 52%, respectively, indicating that hyperammonemia impairs activation of guanylate cyclase. This enzyme is also present in lymphocytes. Activation of guanylate cyclase by SNAP or YC-1 was impaired in lymphocytes from hyperammonemic rats. These results suggest that determination of the activation of soluble guanylate cyclase in lymphocytes could serve as a peripheral marker for impairment of the neuronal glutamate-nitric oxide-cGMP pathway in brain.

Ammonia↗
PubMed1999

Chronic exposure to aluminium impairs the glutamate-nitric oxide-cyclic GMP pathway in the rat in vivo.

Aluminium is neurotoxic and is considered a possible etiologic factor in Alzheimer's disease, dialysis syndrome and other neurological disorders. The molecular mechanism of aluminium-induced impairment of neurological functions remains unclear. We showed that aluminium impairs the glutamate-nitric oxide-cGMP pathway in cultured neurons. The aim of this work was to assess by in vivo brain microdialysis whether chronic administration of aluminium in the drinking water (2.5% aluminium sulfate) also impairs the glutamate-nitric oxide-cGMP pathway in the cerebellum of rats in vivo. Chronic exposure to aluminium reduced NMDA-induced increase of extracellular cGMP by ca 50%. The increase in extracellular cGMP induced by the nitric oxide generating agent S-nitroso-N-acetylpenicillamine was higher (240%) in rats treated with aluminium than in controls. Immunoblotting experiments showed that aluminium reduced the cerebellar content of calmodulin and nitric oxide synthase by 34 and 15%, respectively. Basal activity of soluble guanylate cyclase was decreased by 66% in aluminium-treated rats, while the activity after stimulation with S-nitroso-N-acetylpenicillamine was similar to controls. Basal cGMP in the cerebellar extracellular space was decreased by 50% in aluminium-treated rats. These results indicate that chronic exposure to aluminium reduces the basal activity of guanylate cyclase and impairs the glutamate-nitric oxide-cGMP pathway in the animal in vivo.

Aluminum↗
PubMed1999

Blocking NMDA receptors prevents the oxidative stress induced by acute ammonia intoxication.

Acute ammonia intoxication diminishes the activities of antioxidant enzymes and increases superoxide formation in brain. These effects could play a role in the mechanism of ammonia toxicity. It has been shown that ammonia toxicity is mediated by activation of NMDA receptors. The aim of this work was to assess whether ammonia-induced changes in antioxidant enzymes and in superoxide formation are mediated by activation of NMDA receptors. It is shown that MK-801, an antagonist of NMDA receptors prevents ammonia-induced changes in superoxide dismutase, glutathione peroxidase and catalase. Ammonia intoxication also induces a depletion of glutathione and an increase in lipid peroxidation. Both effects, as well as ammonia-induced increase in superoxide formation are prevented by MK-801. These results indicate that ammonia-induced oxidative stress in brain is mediated by excessive activation of NMDA receptors and support the idea that oxidative stress can play a role in the mechanism of ammonia toxicity.

Acute Disease↗
PubMed1999

Prenatal exposure to aluminum reduces expression of neuronal nitric oxide synthase and of soluble guanylate cyclase and impairs glutamatergic neurotransmission in rat cerebellum.

Exposure to aluminum (Al) produces neurotoxic effects in humans. However, the molecular mechanism of Al neurotoxicity remains unknown. Al interferes with glutamatergic neurotransmission and impairs the neuronal glutamate-nitric oxide-cyclic GMP (cGMP) pathway, especially in rats prenatally exposed to Al. The aim of this work was to assess whether Al interferes with processes associated with activation of NMDA receptors and to study the molecular basis for the Al-induced impairment of the glutamate-nitric oxide-cGMP pathway. We used primary cultures of cerebellar neurons prepared from control rats or from rats prenatally exposed to Al. Prenatal exposure to Al prevented glutamate-induced proteolysis of the microtubule-associated protein-2, disaggregation of microtubules, and neuronal death, indicating an impairment of NMDA receptor-associated signal transduction pathways. Prenatal exposure to Al reduced significantly the content of nitric oxide synthase and guanylate cyclase and increased the content of calmodulin both in cultured neurons and in the whole cerebellum. This effect was selective for proteins of the glutamate-nitric oxide-cGMP pathway as the content of mitogen-activated protein kinase and the synthesis of most proteins were not affected by prenatal exposure to Al. The alterations in the expression of proteins of the glutamate-nitric oxide-cGMP pathway could be responsible for some of the neurotoxic effects of Al.

Aluminum↗
PubMed1998

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.

Animals↗
PubMed1998

Nitroarginine, an inhibitor of nitric oxide synthase, prevents changes in superoxide radical and antioxidant enzymes induced by ammonia intoxication.

Injection of large doses of ammonium salts leads to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We reported that injecting ammonium acetate (7 mmol/kg) to rats increases the production of superoxide and reduces the activities of some antioxidant enzymes in rat liver and brain. We proposed that these effects induced by ammonia intoxication would be mediated by formation of nitric oxide. To test this possibility we tested whether injection of nitroarginine, an inhibitor of nitric oxide synthase, prevents the effects of ammonia intoxication on antioxidant enzymes and superoxide formation. Following injection of ammonia, glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver by 42%, 54% and 44%, respectively. In brain these activities were reduced by 35%, 46% and 65%, respectively. Glutathione reductase remained unchanged. Superoxide production in submitochondrial particles from liver and brain was increased by more than 100% in both tissues. Both reduction of activity of antioxidant enzymes and increased superoxide radical production were prevented by previous injection of 45 mg/kg of nitroarginine, indicating that ammonia induces increased formation of nitric oxide, which in turn reduces the activity of antioxidant enzymes, leading to increased formation of superoxide.

Ammonia↗
PubMed1998

Carnitine inhibits hydrolysis of inositol phospholipids induced by activation of metabotropic receptors.

We previously found that carnitine prevents glutamate neurotoxicity and that this effect is mediated by activation of metabotropic glutamate receptors. We show now that carnitine inhibits the hydrolysis of inositol phospholipids induced by different agonists of metabotropic glutamate receptors (tACPD; (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid; DHPG, (R,S)-3,5-dyhydroxyphenylglycine or S4C3HPG, (S)-4-carboxy-3-hydroxyphenylglycine). The EC50 was ca. 170 microM and the inhibition was complete at 1 mM carnitine. Carnitine also inhibits completely hydrolysis of inositol phospholipids induced by arterenol (agonist of adrenoceptors) and only partially (ca. 50%) that induced by carbachol (agonist of muscarinic receptors). Carnitine did not inhibit phospholipase C activity but inhibits partially (43%) the hydrolysis of inositol phospholipids induced by direct activation of G proteins with AIF4-. The results reported indicate that carnitine inhibits the hydrolysis of inositol phospholipids induced by activation of metabotropic receptors likely by interfering the function of some types of G proteins.

Animals↗
PubMed1998

Carbachol-induced hydrolysis of phospholipids in hippocampal slices may be mediated in part by subsequent activation of metabotropic glutamate receptors.

We observed that AP-3, an antagonist of metabotropic glutamate receptors, reduced carbachol-induced hydrolysis of phospholipids in hippocampal slices. This inhibition could be explained in different ways, e.g.: 1) AP-3 acts also as antagonist of muscarinic receptors mediating the hydrolysis of phospholipids induced by carbachol, 2) Carbachol induces the release of glutamate which, by activating metabotropic glutamate receptors, leads to additional hydrolysis of phospholipids. The aim of this work was to test these possibilities. It is shown that AP-3 reduces carbachol-induced hydrolysis of phospholipids in hippocampal slices but not in cerebellar neurons at 10-14 days of culture, when these cells are not able to induce hydrolysis of phospholipids following activation of metabotropic glutamate receptors. It is also shown that carbachol induces a release of [3H]aspartate in hippocampal slices. The results reported suggest that the hydrolysis of phospholipids induced by carbachol in hippocampal slices would have two components. One part would be due to direct activation by carbachol of muscarinic receptors associated to activation of phospholipase C. This part would not be inhibited by AP-3. The second part would be due to subsequent release of glutamate and activation of metabotropic glutamate receptors. This part would be inhibited by AP-3.

Alanine↗
PubMed1998

Chronic hyperammonemia impairs the glutamate-nitric oxide-cyclic GMP pathway in cerebellar neurons in culture and in the rat in vivo.

The aim of this work was to assess whether ammonia concentrations similar to the increase found in the brain of hyperammonemic rats (100 microM), impair N-methyl-D-aspartate (NMDA) receptor-mediated signal transduction. We first measured glutamate neurotoxicity, which in these neurons is mediated by activation of NMDA receptors, as an initial parameter reflecting activation of NMDA receptor-mediated pathways. Long-term treatment of cultured neurons with ammonia prevents glutamate-induced neuronal death. The EC50 was 20 microM, and at 100 microM the protection was complete. The induction of the protective effect was not immediate, but took several hours. Treatment with 100 microM ammonia did not prevent a glutamate- or NMDA-induced rise of intracellular calcium. Ammonia impaired the glutamate-nitric oxide-cGMP (3',5'-cyclic guanosine monophosphate) pathway in a dose- and time-dependent manner. Glutamate-induced formation of cGMP was reduced by 42%, while activation of nitric oxide synthase was not affected. Ammonia reduced by 31% cGMP formation induced by S-nitroso-N-acetyl-penicillamine (SNAP), a NO-generating agent, confirming that the interference occurs at the level of guanylate cyclase activation by nitric oxide. To assess whether chronic moderate hyperammonemia in vivo also impairs the glutamate-nitric oxide-cGMP pathway, we determined by in vivo brain microdialysis in freely moving rats the formation of cGMP induced by NMDA. In hyperammonemic rats, the formation of cGMP induced by NMDA and SNAP was reduced by ca. 60 and 41%, respectively, indicating that chronic hyperammonemia in the animal in vivo also impairs the glutamate-nitric oxide-cGMP pathway. Impairment of this pathway can contribute to the neurological alterations found in hyperammonemia and hepatic encephalopathy.

Ammonia↗
PubMed1998

Chronic exposure to aluminum impairs neuronal glutamate-nitric oxide-cyclic GMP pathway.

Humans are exposed to aluminum from environmental sources and therapeutic treatments. However, aluminum is neurotoxic and is considered a possible etiologic factor in Alzheimer's disease and other neurological disorders. The molecular mechanism of aluminum neurotoxicity is not understood. We tested the effects of aluminum on the glutamate-nitric oxide-cyclic GMP pathway in cultured neurons. Neurons were exposed to 50 microM aluminum in culture medium for short-term (4 h) or long-term (8-14 days) periods, or rats were prenatally exposed, i.e., 3.7% aluminum sulfate in the drinking water, during gestation. Chronic (but not short-term) exposure of neurons to aluminum decreased glutamate-induced activation of nitric oxide synthase by 38% and the formation of cyclic GMP by 77%. The formation of cyclic GMP induced by the nitric oxide-generating agent S-nitroso-N-acetylpenicillamine was reduced by 33%. In neurons from rats prenatally exposed to aluminum but not exposed to it during culture, glutamate-induced formation of cyclic GMP was inhibited by 81%, and activation of nitric oxide synthase was decreased by 85%. The formation of cyclic GMP induced by S-nitroso-N-acetylpenicillamine was not affected. These results indicate that chronic exposure to aluminum impairs glutamate-induced activation of nitric oxide synthase and nitric oxide-induced activation of guanylate cyclase. Impairment of the glutamate-nitric oxide-cyclic GMP pathway in neurons may contribute to aluminum neurotoxicity.

Aluminum↗
PubMed1997

Diet induced hyperammonemia decreases neuronal nuclear size in rat entorhinal cortex.

Hepatic encephalopathy is mainly caused by an excess of ammonium ions. Among other effects, glutamate transmission in the brain is impaired, and thereof, neuronal function in multiple systems is affected. We investigated in rats the effect of diet induced hyperammonemia in the entorhinal cortex, a well known glutamatergic pathway to the dentate gyrus, by measuring the neuronal nuclear area in two entorhinal cortex subfields (dorsolateral subfield (DLE) and dorsal intermediate subfield (DIE); [Insausti, R., Herrero, M.T. and Witter, M.P., Origin and distribution of cortical efferents from the entorhinal cortex in the rat, Hippocampus, 7 (1997) 146-183]) that project to separate septotemporal levels of the hippocampus. After 2, and more overtly, after 8 weeks of the ammonium enriched diet consumption, the neuronal nuclear size in layers II, III, V and VI of both entorhinal cortex subfields showed a significant reduction in size. We conclude that already at 2 weeks of treatment there is a decrease in neuronal nuclear size in all layers of the entorhinal cortex, which might have widespread functional effects on cortical and subcortical structures.

Acetates↗
PubMed1997

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. In brain mitochondria isolated from ammonia-resistant animals, the ammonia-induced effect on such State 3 rates was not observed. In brain mitochondria from hyperammonemic rats without convulsions, a small increase in the activity of malate dehydrogenase was observed; glutamate dehydrogenase, succinate dehydrogenase, and aspartate aminotransferase were not affected. In brain mitochondria from rats with ammonia-induced convulsions, the activities of malate dehydrogenase and succinate dehydrogenase were reduced significantly. Ammonium acetate injection to rats was associated with a 5-fold increase in the brain mitochondrial ammonium ion content and a decrease (ca. 50%) in brain mitochondrial glutamate and aspartate; brain mitochondrial malate and 2-oxoglutarate levels remained unchanged. The rate of the malate-aspartate shuttle in brain mitochondria of hyperammonemic rats was decreased by 20% as compared to corresponding rate in control rats. We conclude that acute administration of ammonium acetate induces serious disturbances in the electron-transport chain, interferences of the malate-aspartate shuttle, alterations of the levels of shuttle intermediates and inhibition of the activities of malate and succinate dehydrogenases in brain mitochondria.

Acetates↗
PubMed1997

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. This is accomplished by treatment with a detergent solution, the somatic cell releasing reagent from Sigma. This reagent is claimed by the manufacturer to release ATP from a suspension of viable somatic cells. The procedure has been adapted to be used for attached cells (neurons or astrocytes growing in monolayer), thus avoiding the use of alternative time-consuming procedures to release ATP such as boiling buffers or trichloroacetic acid. After its release the free ATP was measured using the firefly luciferase reaction. We have used this protocol to assess the effect of neurotoxic concentrations of glutamate on the intracellular content of ATP in neurons. The same procedure has been used successfully to determine intracellular ATP in primary cultures of astrocytes.

Adenosine Triphosphate↗
PubMed1997

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. 2. As glutamine synthetase in brain is located mainly in astrocytes, we used primary cultures of astrocytes to assess the effects of increasing or decreasing nitric oxide levels on glutamine synthesis in intact astrocytes. 3. Nitric oxide levels were decreased by adding nitroarginine, an inhibitor of nitric oxide synthase. To increase nitric oxide we used S-nitroso-N-acetylpenicillamine, a nitric oxide generating agent. 4. It is shown that S-nitroso-N-acetylpenicillamine decreases glutamine synthesis in intact astrocytes by approximately 40-50%. Nitroarginine increases glutamine synthesis slightly in intact astrocytes. 5. These results indicate that brain glutamine synthesis may be modulated in vivo by nitric oxide.

Animals↗
PubMed1997

Superoxide production and antioxidant enzymes in ammonia intoxication in rats.

Injection of large doses of ammonium salts lead to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We have tested the effect of injecting 7 mmol/kg of ammonium acetate on the production of superoxide and on the activities of some antioxidant enzymes in rat liver, brain, erythrocytes and plasma. Glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver and brain (both in cytosolic and mitochondrial fractions) and also in blood red cells, while glutathione reductase activity remained unchanged. Superoxide production in submitochondrial particles from liver and brain was increased by more than 100% in both tissues. Both diminished activity of antioxidant enzymes and increased superoxide radical production could lead to oxidative stress and cell damage, which could be involved in the mechanism of acute ammonia toxicity.

Ammonia↗
PubMed1997

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. It is shown that glutamate and N-methyl-D-aspartate neurotoxicity are prevented by the following compounds: 1-amino-cyclo-pentane-trans-1,3-dicarboxylic acid, agonist of mGluR1, 2, 3 and 5; 3,5-dihydroxyphenylglycine, agonist of mGluR1 and 5; S-4-carboxy-3-hydroxyphenylglycine, agonist of mGluR5 and antagonist of mGluR1; trans-azetidine-2,4-dicarboxylic acid, agonist of mGluR5. Glutamate neurotoxicity is not prevented by (2S,1'S,2'S)-2(2'-carboxycyclopropyl)glycine, an agonist of mGluR2 and mGluR3. Moreover, the protective effect of 1-aminocyclo-pentane-trans-1,3-dicarboxylic acid is prevented by alpha-methyl-4-carboxyphenylglycine, an antagonist of mGluR1 and 5, but not by alpha-methyl-4-tetrazoylphenylglycine, an antagonist of mGluR2 and 3. A protective effect of activation of mGluR1 can not be ruled out because of the limitations imposed by the lack of specificity of the agonists and antagonists currently available. The results shown clearly indicate that activation of mGluR5 prevents glutamate and N-methyl-D-aspartate neurotoxicity in primary cultures of cerebellar neurons.

Animals↗