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V Felipo

Publications and source records attributed to V Felipo.

At least 19 recordsLinked to original sources
PubMed1996

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). PMA-induced phosphorylation of these proteins is prevented by H7. When quiescent Neuro 2a were stimulated to proliferate by addition of serum, Raf and MAP kinase were rapidly phosphorylated. Serum-induced phosphorylation of Raf and MAP kinase is prevented by H7. These results suggest that, in Neuro 2a cells, the control of proliferation by protein kinase C could be mediated by phosphorylation (and concomitant activation) of Raf and MAP kinase.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

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.

Ammonia↗
PubMed1996

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. All treatments reduced significantly (50-70%) the content of PKC alpha. None of the treatments altered PKC zeta or epsilon. The content of PKC delta was increased (88-120%) in cells treated with PKC inhibitors but was slightly reduced in cells incubated in medium without glutamine. However, none of the treatments affected the content of the corresponding mRNAs. Long-term treatment of synchronized cells with the phorbol ester PMA depletes PKC alpha but not PKC delta or zeta and only partially PKC epsilon. This treatment with PMA did not affect DNA synthesis, indicating that PKC alpha does not play a significant role in the control of proliferation of these cells.

Animals↗
PubMed1996

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. 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 microM and peaked (85%) at 100 microM. 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.

Animals↗
PubMed1996

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. It is shown that betaine, trimethylamine-N-oxide, choline, acetylcholine, carbachol and acetylcarnitine prevent ammonia toxicity in mice. They also prevent glutamate but not N-methyl-D-aspartate neurotoxicity. Choline, acetylcholine and acetylcarnitine afford partial (approximately 50%) protection at nanomolar concentrations and nearly complete protection at micromolar concentrations. Trimethylamine-N-oxide, carbachol and betaine afford nearly complete protection at approximately 0.2 mM. The protective effect against glutamate neurotoxicity is prevented by 2-amino-3-phosphonopropionic acid, an antagonist of metabotropic glutamate receptors. Atropine, an antagonist of muscarinic receptors, prevents the protective effect of most of the above compounds against ammonia toxicity in mice and against glutamate toxicity in cultured neurons. These results support the idea that acute ammonia toxicity is mediated by activation of N-methyl-D-aspartate receptors and that glutamate neurotoxicity could be prevented by activating metabotropic glutamate receptors and/or muscarinic receptors.

Ammonia↗
PubMed1995

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.

Ammonia↗
PubMed1995

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%. Similar effects were induced by glutamate, NMDA and kainate while quisqualate, AMPA or trans-ACPD did not affect significantly ATP content. The EC50 were approximately 6, 25 and 30 microM for glutamate, NMDA and kainate, respectively. DNQX and AP-5, competitive antagonists of kainate and NMDA receptors, respectively, prevented in a dose-dependent manner the glutamate-induced depletion of ATP. These results indicate that glutamate-induced depletion of ATP is mediated by activation of kainate and NMDA receptors. Glutamate-induced neuronal death was prevented by MK-801, calphostin C, H7, carnitine, nitroarginine and W7. However, only MK-801 and W7 prevented glutamate-induced depletion of ATP, while calphostin C, H7, carnitine and nitroarginine did not. This indicates that there is not a direct correlation between ATP depletion and neuronal death.

Adenosine Triphosphate↗
PubMed1995

Alcohol exposure during brain development reduces 3H-MK-801 binding and enhances metabotropic-glutamate receptor-stimulated phosphoinositide hydrolysis in rat hippocampus.

Glutamate receptors play important roles during brain development. We have investigated the effect of chronic maternal alcohol intake on the ontogenic profile of hippocampal glutamate receptor subtypes in their offspring. Binding of 3H-MK-801 to N-methyl-D-aspartate (NMDA) receptor was measured in isolated membranes from the hippocampus of the offspring of pair-fed control and alcohol-fed rats at different times during the postnatal life. Phosphatidylinositol triphosphate (PIP2) hydrolysis was also assayed to provide a measure of the possible effect of ethanol on the metabotropic glutamate receptor (mGluR). In pair-fed control rats, at postnatal day (PND) 3, the 3H-MK-801 binding represents 60% of adult values. Binding then rises to 170% at PND 11, and gradually decreases to adult levels. A transient overshoot in the mGluR-coupled PIP2 hydrolysis was also observed during postnatal development in rat hippocampus. Alcohol-exposed rats showed a similar pattern, but a significant decrease in the specific binding for NMDA receptor was observed on all the postnatal days analyzed. In addition, alcohol exposure significantly decreases the number of specific 3H-MK-801 binding sites, with no change in the affinity of the sites for 3H-MK-801. Moreover, this treatment enhanced the mGluR-activated PIP2 hydrolysis in hippocampus of alcohol-exposed rats. These results may contribute to an understanding of the toxic effects of ethanol on the developing central nervous system (CNS) and help explain the cognitive deficits associated with prenatal alcohol exposure.

Animals↗
PubMed1995

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 nitroarginine 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 (approximately 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.

Amino Acid Oxidoreductases↗
PubMed1995

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. This treatment resulted in a marked decrease of the growth rate of the animals, which was maintained even 1 month after normalization of ammonia levels. Rats exposed to ammonia were more resistant than controls to acute ammonia toxicity 13 days after feeding a normal diet but not at 3 months. Primary cultures of cerebellar neurons from hyperammonemic rats showed decreased binding of [3H]MK-801 and were remarkably more resistant than controls to glutamate and NMDA toxicities. Also, the increase in aspartate aminotransferase activity induced by low concentrations of NMDA was not produced in such cultures. These results indicate that exposure to ammonia during the prenatal and lactation periods results in long-lasting impairment of NMDA receptor function. This would be the reason for the delayed protection afforded by exposure to low ammonia levels against acute ammonia toxicity in animals and against glutamate and NMDA toxicity in neuronal cultures.

Aging↗
PubMed1994

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. Moreover it appears to be a protease, because aprotinin inhibits ADR activity. ADR activity can be also inhibited by the protein kinase C inhibitors, 1-(5-isoquinoline-sulfonyl)-2- methylpiperazine (H7) and calphostin C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Molecular mechanism of acute ammonia toxicity and of its prevention by L-carnitine.

In summary, we propose that acute ammonia intoxication leads to increased extracellular concentration of glutamate in brain and results in activation of the NMDA receptor. Activation of this receptor mediates ATP depletion and ammonia toxicity since blocking the NMDA receptor with MK-801 prevents both phenomena. Ammonia-induced metabolic alterations (in glycogen, glucose, pyruvate, lactate, glutamine, glutamate, etc) are not prevented by MK-801 and, therefore, it seems that they do not play a direct role in ammonia-induced ATP depletion nor in the molecular mechanism of acute ammonia toxicity. The above results suggest that ammonia-induced ATP depletion is due to activation of Na+/K(+)-ATPase, which, in turn, is a consequence of decreased phosphorylation by protein kinase C. This can be due to decreased activity of PKC or to increased activity of a protein phosphatase. We also show that L-carnitine prevents glutamate toxicity in primary neuronal cultures. The results shown indicate that carnitine increases the affinity of glutamate for the quisqualate type (including metabotropic) of glutamate receptors. Also, blocking the metabotropic receptor with AP-3 prevents the protective effect of L-carnitine, indicating that activation of this receptor mediates the protective effect of carnitine. We suggest that the protective effect of carnitine against acute ammonia toxicity in animals is due to the protection against glutamate neurotoxicity according to the above mechanisms.

Ammonia↗
PubMed1994

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. We have tested this hypothesis using primary cultures of neurons. L-carnitine prevented glutamate neurotoxicity in a dose-dependent manner similar to that required to prevent ammonia toxicity in animals. It is also shown that L-carnitine increases selectively the affinity of glutamate for the quisqualate type of glutamate receptors, while the affinity for the kainate and NMDA receptors is slightly decreased. L-carnitine prevents the increase in cytoplasmic Ca2+ induced by addition of glutamate. The Ca2+ levels rose 4.8-fold following addition of 1 mM glutamate, however, when the neurons were incubated previously with 5 mM L-carnitine, the Ca2+ levels increased only by 50%. Also, AP-3, an antagonist of the metabotropic receptor prevents the protective effect of L-carnitine against glutamate neurotoxicity. We suggest, therefore, that the protective effect of L-carnitine against glutamate toxicity is due to the increased affinity of glutamate for the metabotropic receptor. This mechanism could also explain the protection by L-carnitine against acute ammonia toxicity.

Ammonia↗
PubMed1994

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. It is also shown that ammonium salts added in vitro to the assay mixture inhibit acetylcholinesterase in brain homogenates competitively. The Ki values for inhibition of the enzyme in vitro were 7.2 and 8.5 mM for ammonium acetate and ammonium chloride, respectively, when acetylcholinesterase was assayed in rat brain homogenates, and 7.6 and 8.3 mM when assayed in mice brain homogenates. These results suggest that at least part of the neurologic effects of ammonia could be mediated by an increase of acetylcholine as a consequence of the inhibition of acetylcholinesterase.

Acetates↗
PubMed1994

H7, an inhibitor of protein kinase C, inhibits tumour cell division in mice bearing ascitic Ehrlich's carcinoma.

We have previously shown that H7, an inhibitor of protein kinase C (PKC), inhibits proliferation of several cell lines as well as of primary cultured cells from human tumours. The aim of this work was to assess whether H7 is able to prevent the division of tumour cells in mice bearing Ehrlich's ascitic carcinoma. The LD50 of H7 injected intravenously was 61 mg/kg and 94 mg/kg for starved and fed mice, respectively. Acute intraperitoneal injection of 100 mg/kg of H7 decreased the number of mitoses in tumoral cells from ascitic fluid of mice bearing the carcinoma. The reduction was maximal (approximately 50%) after 90 min and then the number of mitosis rose due to a decrease in H7. Continuous delivery of H7 from mini-osmotic pumps implanted on the backs of the mice reduced the number of mitoses by approximately 65%, and the effect was maintained for approximately 24 h. The effect cannot be maintained for longer because H7 is unstable at body temperature. These results indicate that inhibition of PKC can block division of tumour cells in carcinoma-bearing animals, and support the idea that inhibitors of PKC could be useful for the clinical control of proliferation of certain tumours.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

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. 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.

Acetates↗
PubMed1993

Hyperammonemia decreases protein-kinase-C-dependent phosphorylation of microtubule-associated protein 2 and increases its binding to tubulin.

Hyperammonemia increases the polymerization of brain microtubules, which is controlled by the binding of microtubule-associated protein (MAP) 2; binding of MAP-2 is, in turn, regulated by phosphorylation. We have found that the binding of MAP-2 to tubulin is greatly increased by hyperammonemia, however, the brain content of MAP-2 is not affected. Microtubules isolated from hyperammonemic rats contained approximately twice the MAP-2/mg microtubular protein that of microtubules isolated from control animals. MAP isolated from brain microtubules of hyperammonemic rats stimulated the polymerization of tubulin more than MAP isolated from control animals. This appears to be due to the increased content of MAP-2. In vitro phosphorylation, using brain homogenates, showed that protein-kinase-C-dependent phosphorylation of MAP-2 was markedly decreased in hyperammonemic rats. Hyperammonemia also affected the intracellular distribution of brain protein kinase C; its content in the cytosol increased about 23%, while in membranes it decreased by 46%. The possible role of decreased protein-kinase-C-dependent phosphorylation on the increased binding of MAP-2 to tubulin and in the increased polymerization of microtubules in the brain of hyperammonemic rats is discussed.

Ammonia↗