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.     

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.     

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.     

Molecular pharmacology of human NMDA receptors

N-methyl-d-aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission. NMDA receptors are also important drug targets that are implicated in a number of pathophysiological conditions. To facilitate the transition from lead compounds in pre-clinical animal models to drug candidates for human use, it is important to establish whether NMDA receptor ligands have similar properties at rodent and human NMDA receptors. Here, we compare amino acid sequences for human and rat NMDA receptor subunits and discuss inter-species variation in the context of our current knowledge of the relationship between NMDA receptor structure and function. We summarize studies on the biophysical properties of human NMDA receptors and compare these properties to those of rat orthologs. Finally, we provide a comprehensive pharmacological characterization that allows side-by-side comparison of agonists, un-competitive antagonists, GluN2B-selective non-competitive antagonists, and GluN2C/D-selective modulators at recombinant human and rat NMDA receptors. The evaluation of biophysical properties and pharmacological probes acting at different sites on the receptor suggest that the binding sites and conformational changes leading to channel gating in response to agonist binding are highly conserved between human and rat NMDA receptors. In summary, the results of this study suggest that no major detectable differences exist in the pharmacological and functional properties of human and rat NMDA receptors.     

Effects of ammonia and allopurinol on rat hippocampal NMDA receptors

Ammonia is considered to be the main agent responsible for hepatic encephalopathy which progressively leads to altered mental status. N‐methyl‐D‐aspartate (NMDA) is an ionotropic glutamate receptor, which is involved in synaptogenesis, memory and neurotoxicity. The aim of this study was to investigate the effects of ammonia intoxication and allopurinol, a xanthine oxidase (XO) inhibitor, on NMDA receptor subunits, NR2A and NR2B, in the hippocampus of rats. Thirty‐six male rats were divided into three groups (n = 12/group) as follows: (1)control group (phosphate buffered saline (PBS) solution); (2)ammonia group (ammonium acetate, 2.5 mmol/kg), (3)ammonia + allopurinol group (ammonium acetate, 2.5 mmol/kg, allopurinol, 50 mg/kg). Each rat received intraperitoneal injection for 28 days. Western Blotting technique was used for detecting NR2A and NR2B expressions. Both NR2A and NR2B subunit expressions decreased 27 and 11%, respectively, in ammonia group with respect to the control group. Ammonium acetate decreased significantly in NR2A subunit expressions in the hippocampus (p < 0.01). Administration of ammonia + allopurinol caused statistically significant increases in NR2A subunit expressions compared to the ammonia group (p < 0.001). The down‐regulation of NMDA receptors caused by ammonium acetate suggest that these receptors may play role in the process of hepatic encephalopathy and using allopurinol may have some protective effects in ammonia toxicity. Copyright © 2010 John Wiley & Sons, Ltd.     

Magnetic resonance analysis of the effects of acute ammonia intoxication on rat brain. Role of NMDA receptors

Acute ammonia intoxication leads to rapid death, which is prevented by blocking N -methyl- d -aspartate (NMDA) receptors. The subsequent mechanisms leading to death remain unclear. Brain edema seems an important step. The aim of this work was to study the effects of acute ammonia intoxication on different cerebral parameters in vivo using magnetic resonance and to assess which effects are mediated by NMDA receptors activation. To assess edema induction, we injected rats with ammonium acetate and measured apparent diffusion coefficient (ADC) in 16 brain areas. We also analyzed the effects on T1, T2, and T2* maps and whether these effects are prevented by blocking NMDA receptors. The effects of acute ammonia intoxication are different in different brain areas. T1 relaxation time is reduced in eight areas. T2 relaxation time is reduced only in ventral thalamus and globus pallidus. ADC values increased in hippocampus, caudate-putamen, substantia nigra and cerebellar cortex, reflecting vasogenic edema. ADC decreased in hypothalamus, reflecting cytotoxic edema. Myo-inositol increased in cerebellum and substantia nigra, reflecting vasogenic edema. N -acetyl-aspartate decreased in cerebellum, reflecting neuronal damage. Changes in N -acetyl-aspartate, T1 and T2 are prevented by blocking NMDA receptors with MK-801 while changes in ADC or myo-inositol (induction of edema) are not.     

Molecular interaction in the mouse PAG between NMDA and opioid receptors in morphine-induced acute thermal nociception

Previous evidence demonstrates that low dose morphine systemic administration induces acute thermal hyperalgesia in normal mice through μOR stimulation of the inositol signaling pathway. We investigated the site of action of morphine and the mechanism of action of μOR activation by morphine to NMDA receptor as it relates to acute thermal hyperalgesia. Our experiments show that acute thermal hyperalgesia is blocked in periaqueductal gray with the μOR antagonist CTOP, the NMDA antagonist MK801 and the protein kinase C inhibitor chelerythrine. Therefore, a site of action of systemically administered morphine low dose on acute thermal hyperalgesic response appears to be located at the periaqueductal gray. At this supraspinal site, μOR stimulation by systemically morphine low dose administration leads to an increased phosphorylation of specific subunit of NMDA receptor. Our experiments show that the phosphorylation of subunit 1 of NMDA receptor parallels the acute thermal hyperalgesia suggesting a role for this subunit in morphine-induced hyperalgesia. Protein kinase C appears to be the key element that links μOR activation by morphine administration to mice with the recruitment of the NMDA/glutamatergic system involved in the thermal hyperalgesic response.     

The effects of isoniazid on hippocampal NMDA receptors: Protective role of Erdosteine

Isoniazid (INH) has neurotoxic effects such as seizure, poor concentration, subtle reduction in memory, anxiety, depression and psychosis. INH-induced toxic effects are thought to be through increased oxidative stress, and these effects have been shown to be prevented by antioxidant therapies in various organs. Increased oxidative stress may be playing a role in these neurotoxic effects. N-methyl D-aspartat receptors (NMDA) are a member of the ionotropic group of glutamate receptors. These receptors are involved in a wide variety of processes in the central nervous system including synaptogenesis, synaptic plasticity, memory and learning. Erdosteine is a potent antioxidant and mucolytic agent. We aimed to investigate adverse effects of INH on rat hippocampal NMDAR receptors, and to elucidate whether erdosteine prevents possible adverse effects of INH. In the present study, compared to control group, NMDAR2A (NR2A) receptors were significantly decreased and malondialdehyde (MDA), end product of lipid peroxidation, production was significantly increased in INH-treated group. On the other hand, administration of erdosteine to INH-treated group significantly increased NR2A receptors and decreased MDA production. In conclusion, decreasing NR2A receptors in hippocampus and increasing lipid peroxidation correlates with the degree of oxidative effects of INH and erdosteine protects above effect of INH on NR2A receptors and membrane damage due to lipid peroxidation by its antioxidant properties.     

Nicotine improves ethanol-induced memory impairment: The role of dorsal hippocampal NMDA receptors

AimsThe current study was undertaken to determine the role of dorsal hippocampal N-methyl-d-aspartate (NMDA) receptors in nicotine's effect on impairment of memory by ethanol.Main methodsAdult male mice were cannulated in the CA1 regions of dorsal hippocampi and trained on a passive avoidance learning task for memory assessment.Key findingsWe found that pre-training intraperitoneal (i.p.) administration of ethanol (0.5 and 1 g/kg) decreased memory retrieval when tested 24 h later. Pre-test administration of ethanol reversed the decrease in inhibitory avoidance response induced by pre-training ethanol. Similar to ethanol, pre-test administration of nicotine (0.125–0.75 mg/kg, s.c.) prevented impairment of memory by pre-training ethanol. In the animals that received ethanol (1 g/kg, i.p) before training and tested following intra-CA1 administration of different doses of NMDA (0.0005–0.005 µg/mouse), no significant change was observed in the retrieval latencies. Co-administration of the same doses of NMDA with an ineffective dose of nicotine (0.125 mg/kg, s.c.) significantly improved the memory retrieval and mimicked the effects of pre-test administration of a higher dose of nicotine. Pre-test intra-CA1 microinjection of MK-801 (0.25–1 µg/mouse), which had no effect alone, in combination with an effective dose of nicotine (0.75 mg/kg, s.c.) prevented the improving effect of nicotine on memory impaired by pre-training ethanol. Moreover, intra-CA1 microinjection of MK-801 reversed the NMDA-induced potentiation of the nicotine response.SignificanceThe results suggest the importance of NMDA glutamate system(s) in the CA1 regions of dorsal hippocampus for improving the effect of nicotine on the ethanol-induced amnesia.     

Characterization of N-methyl-D-aspartate receptor subunits involved in acute ammonia toxicity

Rapid administration of large doses of ammonia leads to death of animals, which is largely prevented by pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists. The present study focuses on a subunit(s) of NMDA receptor involved in ammonia-induced death by use of NMDA receptor GluRepsilon subunit-deficient (GluRepsilon(-/-)) mice and the selective GluRepsilon2 antagonist CP-101,606. Acute ammonia intoxication was induced in mice (eight per group) by a single intraperitoneal (i.p.) injection of ammonium chloride. Appearance of neurological deteriorations depended on the doses of ammonium chloride injected. While wild-type, GluRepsilon1(-/-), GluRepsilon4(-/-), and GluRepsilon1(-/-)/epsilon4(-/-) mice all died by ammonium chloride at 12 mmol/kg during the first tonic convulsions, two of eight GluRepsilon3(-/-) mice survived. Pretreatment of wild-type mice with CP-101,606 prevented two mice from ammonia-induced death. Pretreatment of GluRepsilon3(-/-) mice with CP-101,606 prevented the death of three mice and prolonged the time of death of non-survivors. Similarly, the neuronal form of nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI) as well as the nonselective NOS inhibitor L-NMMA, but not the inducible NOS inhibitor 1400W, partially prevented the death of mice and prolonged the period of death. Furthermore, ammonium chloride prolonged the increase in intracellular free Ca2+ concentration ([Ca2+]i) and subsequent NO production induced by NMDA in the cerebellum. These results suggest that activation of NMDA receptor containing GluRepsilon2 and GluRepsilon3 subunits and following activation of neuronal NOS are involved in acute ammonia intoxication which leads to death of animals.     

Unique mechanism of action of Alzheimer's drugs on brain nicotinic acetylcholine receptors and NMDA receptors

While a variety of hypotheses have been proposed for the cause of Alzheimer's disease, our knowledge is far from complete to explain the disease making it difficult to develop the methods for treatment. In the brain of Alzheimer's patients, both neuronal nicotinic acetylcholine (nACh) receptors and NMDA receptors are known to be down-regulated. Thus four anticholinesterases have been developed and approved for the treatment in the U.S.A. However, these are not ideal drugs considering their side effects and limited effectiveness. Nefiracetam is being developed for the treatment of Alzheimer's and other patients with dementia, and has unique actions in potentiating the activity of both nACh and NMDA receptors as demonstrated by in vitro patch clamp experiments using rat cortical neurons in primary culture. Nefiracetam potentiated alpha4beta2-like ACh- and NMDA-induced currents at nanomolar concentrations forming bell-shaped dose-response curves with the maximum potentiation occurring at 1 and 10 nM, respectively. Nefiracetam potentiated nACh receptor currents via G(s) proteins, but not G(i)/G(o) proteins, PKA or PKC. Nefiracetam potentiation of NMDA currents occurred via interactions with the glycine binding site of the NMDA receptor. The nefiracetam potentiation of both nACh and NMDA receptors in a potent and efficacious manner is deemed responsible for its cognitive enhancing action.     

New developments on the role of NMDA receptors in Alzheimer's disease

Since the initial findings that NMDA receptors play important roles in cellular models of learning as well as neurotoxicity, abnormal function of this receptor has been considered a potential mechanism in the pathophysiology underlying Alzheimer's disease. Treatment of Alzheimer's disease with an NMDA receptor antagonist began several years ago, with some limited success. More recent mechanistic studies have examined the role of NMDA receptors in the synaptic effects of beta amyloid (Aβ).     

Spermine-induced toxicity in cerebellar granule neurons is independent of its actions at NMDA receptors

The neurotoxic actions of polyamines such as spermine have been linked to their modulation of NMDA receptors, resulting in an excitotoxic cell death. Here, we demonstrate that chronic exposure to the polyamine spermine and acute exposure to the combination of spermine and glutamate result in significant toxicity to primary cultures of cerebellar granule neurons (CGNs). However, in both cases this cell death (a) lacks the characteristic cell swelling associated with the necrotic cell death induced by glutamate and (b) is characterized by the widespread formation of apoptotic nuclei. Whereas dizocilpine (MK-801) blocks the synergistic cell death resulting from acute exposure to spermine plus glutamate, neither MK-801 nor the calcium chelator EGTA appreciably attenuates CGN death resulting from chronic exposure to spermine. Consistent with previous reports, glutamate, both acute and chronic, causes CGN death that is characterized by cell swelling, sensitivity to MK-801 and EGTA, and only small numbers of apoptotic nuclei. Spermine-induced toxicity is not blocked by either the protein synthesis inhibitor cycloheximide or the pancaspase inhibitor tert-butoxycarbonyl-Asp-(O-methyl) fluoromethyl ketone. However, the antioxidant butylated hydroxyanisole is an effective blocker of spermine-induced CGN death, suggesting a free-radical component to this cell death. The intact spermine molecule, rather than a catabolic by-product, is required for cell death because the amine oxidase inhibitors N1,N2-bis(2,3-butadienyl)-1,4-butanediamine and aminoguanidine fail to block this toxicity. Thus, in CGNs, spermine-induced toxicity does not occur by its modulation of NMDA receptors, although, under some circumstances, NMDA receptor activation can modulate spermine-induced toxicity.     

Molecular mechanism and physiological role of pexophagy

Pexophagy is a selective autophagy process wherein damaged and/or superfluous peroxisomes undergo vacuolar degradation. In methylotropic yeasts, where pexophagy has been studied most extensively, this process occurs by either micro- or macropexophagy: processes analogous to micro- and macroautophagy. Recent studies have identified specific factors and illustrated mechanisms involved in pexophagy. Although mechanistically pexophagy relies heavily on the core autophagic machinery, the latest findings about the role of auxiliary pexophagy factors have highlighted specialized membrane structures required for micropexophagy, and shown how cargo selectivity is achieved and how cargo size dictates the requirement for these factors during pexophagy. These insights and additional observations in the literature provide a framework for an understanding of the physiological role(s) of pexophagy.     

Neuromodulatory effect of creatine on extracellular action potentials in rat hippocampus: role of NMDA receptors

The creatine (Cr) and phosphocreatine (PCr) system is essential for the buffering and transport of high-energy phosphates. Although achievements made over the last years have highlighted the important role of creatine in several neurological diseases, the adaptive processes elicited by this guanidino compound in hippocampus are poorly understood. In the present study, we showed that creatine (0.5-25mM) gradually increases the amplitude of first population spike (PS) and elicits secondary PS in stratum radiatum of the CA1 region, in hippocampal slices. Creatine also decreased the intensity of the stimulus to induce PS, when compared with hippocampal slices perfused with artificial cerebrospinal fluid (ACSF). The competitive NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5; 100microM) attenuated creatine-induced increase of amplitude of PS and appearance of secondary PS, providing pharmacological evidence of the involvement of NMDA receptors in the electrophysiological effects of creatine. Accordingly, creatine (0.01-1mM) increased [3H]MK-801 binding to hippocampal membranes by 55%, further indicating that this compound modulates NMDA receptor function. These results implicate the NMDA receptor in amplitude and population spike increase elicited by creatine in hippocampus. Furthermore, these data suggest that this guanidino compound may also play a putative role as a neuromodulator in the brain, and that at least some of its effects may be mediated by an increase in glutamatergic function.     

A mechanism for acute aluminium toxicity in fish

Aluminium is acutely toxic to fish in acid waters. The gill is the principal target organ and death is due to a combination of ionoregulatory, osmoregulatory and respiratory dysfunction. The toxic mechanism has hitherto received little direct consideration and is unknown. In this paper the mechanism of acute aluminium toxicity is approached from a chemical perspective. Symptomatic evidence of toxicity is taken from the literature and combined with our own research to elucidate a biochemically sound model to describe a possible mechanism of acute aluminium toxicity in fish. The proposed model delineates the chemical conditions immediately adjacent to the gill surface and emphasizes their importance in aluminium's toxic mode of action. The mechanism is shown to be bipartite. Aluminium binding to functional groups both apically located at the gill surface and intracellularly located within lamellar epithelial cells disrupts the barrier properties of the gill epithelium. The concomitant iono- and osmoregulatory dysfunction results in accelerated cell necrosis, sloughing and death of the fish. The mechanism of epithelial cell death is proposed as a general mechanism of aluminium-induced accelerated cell death.     

The role of NMDA receptors in the slow neuronal degeneration of Parkinson's disease

Summary Parkinson's disease is a disorder, in which neurons of various neuronal systems degenerate. Furthermore, in such degenerating neurons, the cytoskeleton seems to be affected. In this respect, Parkinson's disease resembles Alzheimer's disease. Since it has been shown, that elevated levels of intracellular calcium can disrupt the cytoskeleton and that the stimulation of glutamate (NMDA) receptors can cause high intracellular concentrations of calcium, it has been suggested, that the stimulation of glutamate receptors plays a role in the slow degeneration in Alzheimer's and Parkinson's disease. In case of the degeneration of the dopaminergic nigrostriatal system in Parkinson's disease, neurons that contain calcium binding protein appear to be less vulnerable than the neurons that lack it, suggesting that calcium binding protein might protect these neurons from degeneration by preventing that cytosolic calcium concentrations increase excessively. And, since there is in the nigrostriatal system a glutamatergic afferent pathway (the prefrontonigral projection) and since dopaminergic nigrostriatal neurons contain postsynaptic NMDA receptors, glutamatergic excitation may play a role in the degeneration of the nigrostriatal system in Parkinson's disease. If so, it may be possible to protect the neurodegeneration of these dopaminergic neurons by NMDA receptor antagonists.