Mitochondrial involvement in nitric oxide‐induced cellular death in cortical neurons in culture

Abstract

Nitric oxide (NO) is an unstable molecule with physiological and pathological properties. In brain, NO acts as a modulator of neurotransmission as well as a protector against neuronal death from several death stimuli. However, beside this protector effect, high NO concentrations produce neuronal death by a mechanism in which the caspase pathway is implicated. In this work, we demonstrate that in cortical neurons the NO toxicity is mediated by mitochondrial dysfunction. SNAP, an NO donor, induces apoptosis in these cells because it 1) increases the p53 and 2) induces cytochrome c release and activation of caspase-9 and caspase-3. SNAP also induces necrosis, through 1) breakdown of the mitochondrial membrane potential, 2) ATP decrease, 3) ROS formation, and 4) LDH and ATP release, indicative of oxidative stress and death by necrosis. To sum up, in cortical neurons, high NO concentrations produced cellular death by both an apoptotic and a necrotic mechanism in which the mitochondria are implicated.

Added after Anoxia-Reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons

“…Brain neurons were obtained as described in [25]. Isolated neurons were suspended in EMEM medium containing 0.3 g/l glutamine, 3 g/l glucose, 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.…”

“…Cells were used after 7 days of culture. Cell purity was checked by both, cells staining with cresyl violet to identify neurons, and immunocytochemistry with the specific anti-GFAP antibody [25] to identify glial cells. Under these conditions, the glial cell number in the cultures was of 8.3 ± 3.6% of the total cell population (neurons + glial cells).…”

“…Measurements were preformed as described [25]. Briefly, control and treated neurones (2 × 10 5 ) were incubated with the XTT solution at 0.3 mg/ml final concentration and the orange formazan dye formed was spectrophotometrically quantified using an ELISA plate reader, at 450 nm (reference 650 nm).…”

“…LDH activity was measured as the rate of decrease of the absorbance at 340 nm, resulting from the oxidation of NADH to NAD + as described [25]. Briefly, the culture medium was collected and the neurones were lysed with 0.1 M Tris-HCl (pH 7.4) containing 0.1% Triton X-100, cell suspension was centrifuged at 13,000 g during 5 minutes and LDH activity measured in both preparations, culture medium and cells supernatant.…”

Nitric oxide in neurodegeneration: potential benefits of non-steroidal anti-inflammatories

“…Studies have demonstrated both neuroprotective [29][30][31] and neurotoxic [32][33][34] effects of NO. Furthermore, in addition to the direct effects of NO on cell survival, it has been established that within cells, NO interacts with superoxide to form peroxynitrite.…”