Implication of Glutamate in the Expression of Inducible Nitric Oxide Synthase After Oxygen and Glucose Deprivation in Rat Forebrain Slices

Abstract

Nitric oxide synthesis by inducible nitric oxide synthase (iNOS) has been postulated to contribute to ischemia-reperfusion neurotoxicity. The expression of this enzyme has been demonstrated in cells present in the postischemic brain. The mechanisms of iNOS expression after cerebral ischemia are a subject of current research. We therefore decided to investigate whether glutamate, which is released in ischemia and is implicated in neurotoxicity, might be involved in the mechanisms by which oxygen and glucose deprivation (OGD) leads to the expression of iNOS in rat forebrain slices. In this model, we have shown previously that 20 min of OGD causes the expression of iNOS. We have now found that the NMDA receptor antagonist MK-801 blocks the expression of iNOS, suggesting that the activation of the NMDA subtype of glutamate receptor is implicated in the mechanisms that lead to the expression of this isoform. Moreover, we have found that glutamate alone could trigger the induction process, as shown by the appearance of a Ca 2ϩ -independent NOS activity and by the detection of iNOS mRNA and protein in slices exposed to glutamate. Glutamate-dependent iNOS expression was concentration-dependent and was blocked by EGTA and by the inhibitors of nuclear factor B (NF-B) activation pyrrolidine dithiocarbamate and MG132. In addition, glutamate induced NF-B translocation to the nucleus, an effect that was inhibited by MG132. Taken together, our data suggest that activation of NMDA receptors by glutamate released in ischemia is involved in the expression of iNOS in rat forebrain slices via a Ca 2ϩ -dependent activation of the transcription factor NF-B. To our knowledge, this is the first report showing an implication of excitatory amino acids in the expression of iNOS caused by ischemia. Key Words: Cerebral ischemia-NMDA-Neurotoxicity-Nuclear factor B.

Chronic Stress and Lithium Treatments Alter Hippocampal Glutamate Uptake and Release in the Rat and Potentiate Necrotic Cellular Death After Oxygen and Glucose Deprivation

“…Rats were decapitated, their hippocampi were quickly dissected out and transverse sections (400 lm) were rapidly obtained using a McIlwain tissue chopper. One slice was placed into each well of a 24-well culture plate and preincubated for 15 min in a modified Krebs-Henseleit solution at 37°C in an atmosphere of 5% CO 2 (95%O 2 / 5%CO 2 ) [33].…”

“…Slices corresponding to the ''ischemic'' experimental group were incubated in a solution without glucose for 60 min in an anaerobic chamber, saturated with N 2 , with temperature kept at 37°C. After this period, the incubation solution was replaced with fresh control medium, and slices were incubated for 3 or 24 h at 378C in an atmosphere of 5% CO 2 (95%O 2 /5%CO 2 ) to simulate a ''reperfusion'' period [33]. Assessment of neural injury-LDH assay: Neural cell injury was quantified by the measurement of lactate dehydrogenase (LDH) released from damaged cells into the extracellular fluid.…”

Tetramethylpyrazine Analogue CXC195 Protects against Cerebral Ischemia/Reperfusion Injury in the Rat by an Antioxidant Action via Inhibition of NADPH Oxidase and iNOS Expression

“…NADPH oxidase (NOX), which catalyzes the generation of superoxide anions (O 2 - ∙), is one of the main contributors to excessive ROS in I/R injury [5,6]. Another important ROS generator is nitric oxide (NO) generated from inducible NO synthase (iNOS), which is induced in models of brain ischemia both in vitro and in vivo [7,8]. NO can react with O 2 - ∙ to produce peroxynitrite (ONOO - ), which is a strong oxidative radical that causes protein nitration and dysfunction [9,10].…”

Ferulic acid inhibits nitric oxide-induced apoptosis by enhancing GABAB1 receptor expression in transient focal cerebral ischemia in rats

“…Excitotoxicity induced by activation of glutamate receptors is the predominant mechanism underlying neuronal apoptosis in stroke [4] . Cerebral ischemic damage is associated with excessive release of excitatory glutamate, which subsequently upregulates not only the expression of neuronal nitric oxide synthase (nNOS), but also inducible nitric oxide synthase (iNOS) [5,6] . Numerous studies [7][8][9][10] have shown that the glutamate receptor-postsynaptic density-95 (PSD-95)-nNOS complex and iNOS produce an excessive amount of nitric oxide (NO) during cerebral I/R injury; NO immediately reacts with superoxide radicals to form peroxynitrite, which subsequently nitrates protein tyrosine residues, generating nitrotyrosine and leading to oxidative/nitrative stress.…”

“…Toxic NO-mediated oxidative/nitrative stress during cerebral I/R injury may contribute to the activation of the apoptotic cascade [11] . In vitro studies [5,12] have demonstrated that the NO-induced apoptotic signaling cascade involves mitogen-activated protein (MAP) kinase-mediated Bax translocation from the cytosol to the mitochondria and subsequent caspase-3 activation in cultured In previous studies, FA reduces cerebral ischemic injury by weakening the expression of PSD-95 in the ischemic area and provides neuroprotection against apoptosis partly via inhibiting intercellular adhesion molecule-1 (ICAM-1) mRNA expression in a transient middle cerebral artery occlusion (MCAo) model [13,14] . In addition, FA inhibits glutamate-induced apoptosis through modulation of the MAP kinase signaling pathway in cultured cortical neurons [15] .…”