Nitric oxide produced by non-motoneuron cells enhances rat embryonic motoneuron sensitivity to excitotoxins: comparison in mixed neuron/glia or purified cultures.
The present study compares the sensitivity to chronic exposure to glutamate agonists of SMI-32-positive rat-derived embryonic motoneurons under both mixed neuron/glia and purified cultures. We found that in spite of a trophic role of glia on cultured motoneurons, SMI-32-positive cells are more sensitive to excitotoxicity in the presence of glia than in purified culture, very likely through nitric oxide released by non-neuronal cells. The rank order of potency for inducing toxicity after 48 h incubation was AMPA>kainate>NMDA, with EC(50): 0.43, 4.9 and 49 microM, respectively, in mixed neuron/glia culture and 14, 32 and 135 microM in purified cultures. The effect of NMDA was dose-dependently potentiated by glycine, with similar potency in the two culture conditions. The effect of agonists was completely antagonized by the specific antagonists CNQX, BNQX and MK801 in both culture conditions. Motoneurons were similarly immunoreactive to NR1 and GluR2 antibodies under both mixed neuron/glia and purified cultures, thus confirming the presence of the calcium-impermeant AMPA receptor subtypes and of the obligatory subunit for NMDA receptors. The effect of kainate in mixed neuron/glia culture was reduced by the addition of 40 microM N-nitro-L-arginine or L-NAME, which shifted the EC(50) to 9 microM. By contrast, L-NAME did not modify the effect of kainic acid in purified cultures. These results suggest that the release of nitric oxide by non-neuronal cells in culture enhances glutamate excitotoxicity in SMI-32-positive cells, and that direct activation of ionotropic glutamate receptors is not enough to explain the mechanism of chronic motoneuron degeneration occurring in vivo in amyotrophic lateral sclerosis (ALS).
Comoletti
D
D
Laboratory of Receptor Pharmacology, Mario Negri Institute for Pharmacological Research, Via Eritrea, 62, 20157 Milan, Italy.
Muzio
V
V
Capobianco
A
A
Ravizza
T
T
Mennini
T
T
D000690:Amyotrophic Lateral Sclerosis / Q000209:etiology / Q000378:metabolism / Q000503:physiopathology; D000818:Animals; D000870:Anterior Horn Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005333:Fetus; D018698:Glutamic Acid / Q000378:metabolism*; D005998:Glycine / Q000494:pharmacology; D007608:Kainic Acid / Q000633:toxicity; D008274:Magnesium / Q000494:pharmacology; D016202:N-Methylaspartate / Q000633:toxicity; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D009498:Neurotoxins / Q000378:metabolism / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000633:toxicity
D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D009498:Neurotoxins; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D009569:Nitric Oxide; D018698:Glutamic Acid; D016202:N-Methylaspartate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D008274:Magnesium; D007608:Kainic Acid; D005998:Glycine