Ammonia Prevents Activation of NMDA Receptors by Glutamate in Rat Cerebellar Neuronal Cultures

Abstract

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.

Chronic and acute ammonia toxicity in mudskippers,Periophthalmodon schlosseriandBoleophthalmus boddaerti: brain ammonia and glutamine contents, and effects of methionine sulfoximine and MK801

“…The magnitude of the increase in arterial ammonia concentrations in patients with acute liver failure predicts the evolution of brain herniation in these patients (Clemmensen et al, 1999). In addition, chronic hyperammonemia (unlike acute ammonia intoxication) leads to a depression of excitatory neurotransmission by impairment of NMDA receptor function and by inhibition of NMDA receptor-mediated signal transduction pathways (Felipo and Butterworth, 2002), as demonstrated by a decrease in binding of 3 H-MK801 to membranes from rat hippocampus (Marcaida et al, 1995).…”

“…On the other hand, it has been demonstrated in rats injected intraperitoneally with CH 3 COONH 4 that an activation of NMDA receptors precedes the increase in extracellular glutamate (Hermenegildo et al, 2000). Thus, acute ammonia intoxication in rats involves the activation of NMDA receptors (Marcaida et al, 1992(Marcaida et al, , 1995, and NMDA receptor activation is likely to be initiated by a depolarization of the neuronal membrane (Sugden and Newsholme, 1975;Fan and Szerb, 1993) instead of an increase in extracellular glutamate (Hermenegildo et al, 2000). When (5R, 10S)-(+)-methyl-10, 11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine hydrogen maleate (MK801; a NMDA receptor antagonist) is injected into rats before the injection of CH 3 COONH 4 , it can delay or eliminate the fatal effects of acute ammonia toxicity (Marcaida et al, 1992;Hermenegildo et al, 1996).…”

Prevention of in Vivo Excitotoxicity by a Family of Trialkylglycines, a Novel Class of Neuroprotectants

“…Ca 2ϩ levels were monitored continuously. Free intracellular calcium was quantified using the confocal laser cytometer as previously described (Marcaida et al, 1995a). Each experiment was repeated at least four times with three different neuronal cultures.…”

A NovelN-Methyl-d-aspartate Receptor Open Channel Blocker with in Vivo Neuroprotectant Activity

“…Cells were washed with Locke's solution and the conditioned medium was added back. Cell viability was measured 24 h later, by staining with fluorescein diacetate and propidium iodide (Molecular Probes, Eugene, OR) as described previously (Marcaida et al, 1995). The percentage of surviving neurons was calculated by assessing the ratio of fluorescein diacetate/propidium iodide (green/red) staining directly under the microscope.…”

“…Changes in intracellular free Ca 2ϩ were monitored in single cerebellar neurons using an ACAS 570 confocal laser cytometer (Meridians Instruments, Okemos, MI) (Marcaida et al, 1995). Primary cultures of cerebellar neurons were prepared as described above using 35-mmdiameter tissue culture dishes.…”