Altered modulation of soluble guanylate cyclase by nitric oxide in patients with liver disease.

The glutamate-nitric oxide-cGMP pathway is impaired in brain in vivo in animal models of chronic moderate hyperammonemia either with or without liver failure. The impairment occurs at the level of activation of soluble guanylate cyclase by nitric oxide (NO). It has been suggested that the impairment of this pathway may be responsible for some of the neurological alterations found in hyperammonemia and hepatic encephalopathy. Soluble guanylate cyclase is also present in lymphocytes. Activation of guanylate cyclase by NO is also altered in lymphocytes from hyperammonemic rats or from rats with portacaval anastomosis. We assessed whether soluble guanylate cyclase activation was also altered in human patients with liver disease. We studied activation of soluble guanylate cyclase in lymphocytes from 77 patients with liver disease and 17 controls. The basal content of cGMP in lymphocytes was decreased both in patients with liver cirrhosis and in patients with chronic hepatitis. In contrast, cGMP concentration was increased in plasma from patients with liver disease. Activation of guanylate cyclase by NO was also altered in liver disease and was higher in lymphocytes from patients with cirrhosis or hepatitis than that in lymphocytes from controls. Successful treatment with interferon of patients with hepatitis C reversed all the above alterations. Altered modulation of soluble guanylate cyclase by NO in liver disease may play a role in the neurological and hemodynamic alterations in these patients.

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.

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.

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.

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.

Neurotoxicity of ammonia and glutamate: molecular mechanisms and prevention.

Ammonia is a main factor in the pathogenesis of hepatic encephalopathy. We found that acute ammonia toxicity is mediated by activation of NMDA receptors. Chronic moderate hyperammonemia prevents acute ammonia toxicity in rats. Chronic exposure of cultured neurons to 1 mM ammonia leads to impaired response of the NMDA receptor to activation by its agonists (due to decreased protein kinase C-mediated phosphorylation) and prevents glutamate (Glu) neurotoxicity. Compounds that prevent ammonia toxicity in mice (e.g. carnitine) also prevent Glu toxicity in cultured neurons. These compounds did not prevent activation of NMDA receptor or the rise of Ca2+. They interfered with subsequent steps in the toxic process. The protective effect of carnitine is mediated by activation of metabotropic Glu receptors. Agonists of mGluRs, especially of mGluR5, prevent Glu toxicity. Agonists of muscarinic receptors also prevent Glu toxicity and there seems to be an interplay between muscarinic and metabotropic Glu receptors in the protective effect. We have tried to identify intracellular events involved in the process of neuronal death. It is known that the rise of Ca2+ is an essential step. Glu leads to depletion of ATP; some compounds (e.g. carnitine) prevent Glu-induced neuronal death without preventing ATP depletion: additional events are required for neuronal death. Glu induces activation of Na+/K+-ATPase, which could be involved in the toxic process. Inhibitors of protein kinase C, calcineurin or nitric oxide synthase prevent Glu toxicity. Our results indicate that Glu toxicity can be prevented at different steps or by activating receptors coupled to the transduction pathways interfering with the toxic process. Agents acting on these steps could prevent excitotoxicity in vivo in animals.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.