No abstract

2 2 heterotetrameric molecule, through its ligand-induced tyrosine kinase activity, leading to the phosphorylation of the cytoplasmic domain of the -subunits (6,7). Receptor activation promotes the rapid phosphorylation on multiple tyrosine residues of insulin receptor substrate (IRS)-1 and I R S-2, which allows the binding, through the phosphorylated sequences, of s r c homology domain 2 (SH2) domains of various proteins, initiating further IGF-I-dependent signaling events (8,9). Among the proteins interacting with IRS-1 and IRS-2 are 1) Grb-2, which activates the Ras/Raf/ERK signaling pathway; 2) Shc, which in addition to the interaction with IRS-1 can be activated directly by phosphorylation through the IGF-I receptor, depending on the cell type; and 3) the p85 subunit of the phosphatidylinositol (PI) 3-kinase (8-10). PI 3-kinase is a family of isoenzymes with dual lipid and serine/threonine kinase activities that acts as a key regulator in a variety of cellular responses, including glucose transport, mitogenesis and differentiation, and membrane dynamics, and as an anti-apoptotic mediator (4,(11)(12)(13)(14). The kinase activity is located in the p110 subunit, whereas p85 acts as the regulatory part of the complex. Interaction between phosphorylated IRS-1 and p85 allows the activation of p110 and the localization of this subunit in the proximity of its substrates near the membrane, among them p85 and IRS-1 (12,14). The signaling downstream PI 3-kinase is a subject of current interest because a large number of targets appear to be modulated through this pathway (13).R e c e n t l y, it has been shown that inhibition of PI 3-kinase leads to an overexpression of nitric oxide synthase (NOS)-2, the enzyme responsible for the high-output NO synthesis in macrophages, microglia, and other cells stimulated with lipopolysaccharide (LPS) or pro-inflammatory cytokines (15,16). Expression of NOS-2 in rodent cells has been studied e x h a u s t i v e l y. The process is regulated through synergistic cooperation between the transcription factors NF-B, Stat-1, and IRF-1, at least in response to LPS and IFN-c h a l l e n g e (17,18). Some data indicate that NF-B activation is a critical event in the expression of NOS-2, and this process depends on the degradation of the corresponding inhibitory proteins

No 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.