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 a-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 nondesensitizing 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.

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

“…To address whether NCX is involved in the initial events of Ca 2 þ influx, we investigated the uptake of 45 Ca 2 þ in cultured hippocampal neurons, following stimulation of AMPA receptors. The excitotoxic paradigm of nondesensitizing AMPA receptor activation extensively studied was used, 11,16,17 since in this model, calpains, but not caspases, are responsible for neuronal death. 11 Hippocampal neurons were treated with kainate (KA) (100 mM) for 5 min, which is not toxic by itself in a short exposure, 17 together with cyclothiazide (CTZ, 30 mM).…”

“…The excitotoxic paradigm of nondesensitizing AMPA receptor activation extensively studied was used, 11,16,17 since in this model, calpains, but not caspases, are responsible for neuronal death. 11 Hippocampal neurons were treated with kainate (KA) (100 mM) for 5 min, which is not toxic by itself in a short exposure, 17 together with cyclothiazide (CTZ, 30 mM). CTZ is a selective blocker of AMPA receptor desensitization, 19 and is a useful tool to selectively unmask effects mediated by AMPA receptors.…”

“…CTZ is a selective blocker of AMPA receptor desensitization, 19 and is a useful tool to selectively unmask effects mediated by AMPA receptors. 16,17,20 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl] isothiourea (KB-R7943) (20 mM) selectively inhibits the reverse mode of NCX and was used for this purpose, being three-fold more effective on NCX3 than on NCX1 or NCX2. 21 Treatment for 5 min with KA plus CTZ increased 45 Ca 2 þ uptake to 17.370.59 nmol Ca 2 þ /10 6 cells (Po0.001; Figure 1a), as compared to non-stimulated hippocampal neurons (1.570.05 nmol Ca 2 þ /10 6 cells).…”

“…14, 15 We recently reported that neurotoxicity induced by activation of AMPA receptors is characterized by calpain activation, lack of caspase activation, nuclear condensation/fragmentation, release of cytochrome c from mitochondria, decreased intracellular ATP levels, production of nitric oxide, moderate superoxide production and increased levels of peroxynitrite. [16][17][18] In this in vitro model of excitotoxicity, the cell …”

“…14, 15 We recently reported that neurotoxicity induced by activation of AMPA receptors is characterized by calpain activation, lack of caspase activation, nuclear condensation/fragmentation, release of cytochrome c from mitochondria, decreased intracellular ATP levels, production of nitric oxide, moderate superoxide production and increased levels of peroxynitrite. [16][17][18] In this in vitro model of excitotoxicity, the cell death mechanisms are mostly dependent on calpains, 11 providing a good model for studying calpain-dependent phenomena. Nevertheless, it is not known whether the influx of Ca 2 þ through reversal of the exchanger is related to activation of calpains in this model, where calpains are the main executioners of cell death, and whether inhibition of this component of Ca 2 þ entry during AMPA receptor activation is neuroprotective.…”