title: Neuronal nitric oxide synthase proteolysis limits the involvement of nitric oxide in kainate-induced neurotoxicity in hippocampal neurons.
abstract: In this work, we investigated the role of nitric oxide (NO) in neurotoxicity triggered by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation in cultured hippocampal neurons. In the presence of cyclothiazide (CTZ), short-term exposures to kainate (KA; 5 and 15 min, followed by 24-h recovery) decreased cell viability. Both NBQX and d-AP-5 decreased the neurotoxicity caused by KA plus CTZ. Long-term exposures to KA plus CTZ (24 h) resulted in increased toxicity. In short-, but not in long-term exposures, the presence of NO synthase (NOS) inhibitors (l-NAME and 7-NI) decreased the toxicity induced by KA plus CTZ. We also found that KA plus CTZ (15-min exposure) significantly increased cGMP levels. Furthermore, short-term exposures lead to decreased intracellular ATP levels, which was prevented by NBQX, d-AP-5 and NOS inhibitors. Immunoblot analysis revealed that KA induced neuronal NOS (nNOS) proteolysis, gradually lowering the levels of nNOS according to the time of exposure. Calpain, but not caspase-3 inhibitors, prevented this effect. Overall, these results show that NO is involved in the neurotoxicity caused by activation of non-desensitizing AMPA receptors, although to a limited extent, since AMPA receptor activation triggers mechanisms that lead to nNOS proteolysis by calpains, preventing a further contribution of NO to the neurotoxic process.
authors.affiliation: Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, Coimbra, Portugal.
authors.forename: Inês M
authors.initials: IM
authors.lastname: Araújo
authors.forename: António F
authors.initials: AF
authors.lastname: Ambrósio
authors.forename: Ermelindo C
authors.initials: EC
authors.lastname: Leal
authors.forename: Paulo F
authors.initials: PF
authors.lastname: Santos
authors.forename: Arsélio P
authors.initials: AP
authors.lastname: Carvalho
authors.forename: Caetana M
authors.initials: CM
authors.lastname: Carvalho
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007608:Kainic Acid / Q000633:toxicity*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010447:Peptide Hydrolases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D013997:Time Factors
chemical_list: D000906:Antibodies; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D010447:Peptide Hydrolases; D002154:Calpain; D006152:Cyclic GMP; D007608:Kainic Acid