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.
J. Neurochem. (2010) 113, 1343–1355.
Abstract
The involvement of plasma membrane glutamate transporters (EAATs – excitatory aminoacid transporters) in the pathophysiology of ischemia has been widely studied, but little is known about the role of vesicular glutamate transporters (VGLUTs) in the ischemic process. We analyzed the expression of VGLUT1–3 in the cortex and caudate‐putamen of rats subjected to transient middle cerebral artery occlusion. Western blot and immunohistochemistry revealed an increase of VGLUT1 signal in cortex and caudate‐putamen until 3 days of reperfusion followed by a reduction 7 days after the ischemic insult. By contrast, VGLUT2 and 3 were drastically reduced. Confocal microscopy revealed an increase in VGLUT2 and 3 immunolabelling in the reactive astrocytes of the ischemic corpus callosum and cortex. Changes in VGLUTs and EAATs expression were differently correlated to neurological deficits. Interestingly, changes in VGLUT1 and EAAT2 expression showed a significant positive correlation in caudate‐putamen. Taken together, these results suggest a contribution of VGLUTs to glutamate release in these structures, which could promote neuroblast migration and neurogenesis during ischemic recovery, and a possible interplay with EAATs in the regulation of glutamate levels, at least in the first stages of ischemic recovery.
Nigella sativa L. (NS) has been used for medicinal purposes since ancient times. This
study aimed to investigate the cytotoxicity of NS dry methanolic extract on cultured
cortical neurons and its influence on neurotransmitter release, as well as the presence of
excitatory (glutamate and aspartate) and inhibitory amino acids (gamma-aminobutyric
acid—GABA—and glycine) in NS extract. Cultured rat cortical neurons were exposed to
different times and concentrations of NS dry methanolic extract and cell viability was
then determined by a quantitative colorimetric method. NS did not induce any toxicity.
The secretion of different amino acids was studied in primary cultured cortical neurons
by high-performance liquid chromatography (HPLC) using a derivation before injection
with dansyl chloride. NS modulated amino acid release in cultured neurons; GABA was
significantly increased whereas secretion of glutamate, aspartate, and glycine were
decreased. The in vitro findings support the hypothesis that the sedative and depressive
effects of NS observed in vivo could be based on changes of inhibitory/excitatory amino
acids levels.
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