Inhibition of phosphatidylinositol 3-kinase induces nitric-oxide synthase in lipopolysaccharide- or cytokine-stimulated C-6 glial cells

Nitric oxide (NO) produced by inducible nitric-oxide synthase (iNOS) in dif ferent cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of demyelinating and neurod egenerative diseases, The present study underlines the importance of phosph atidylinositol 3-kinase (PI 3-kinase) in the expression of iNOS in C-6 glia l cells and rat primary astrocytes. Bacterial lipopolysaccharide (LPS) or i nterleukin-1 beta (IL-1 beta) was unable to induce the expression of iNOS a nd the production of NO in rat C-6 glial cells. Similarly, wortmannin and L Y294002, compounds that inhibit PI3-kinase, were also unable to induce the expression of NOS and the production of NO. However, a combination of wortm annin or LY294002 with LPS or IL-1 beta induced the expression of iNOS and the production of NO in C-6 glial cells. Consistent with the induction of i NOS, wortmannin also induced iNOS promoter-derived chloramphenicol acetyltr ansferase activity in LPS- or IL-1 beta-treated C-6 glial cells. The expres sion of iNOS by LPS in C-6 glial cells expressing a dominant-negative mutan t of p85 alpha, the regulatory subunit of PI 3-kinase, further supports the conclusion that inhibition of PI 3-kinase provides a necessary signal for the induction of iNOS, Next we examined the effect of wortmannin on the act ivation of mitogen-activated protein (MAP) kinase and nuclear factor NF-kap pa B in LPS- or IL-1 beta-stimulated C-6 glial cells. In contrast to the in ability of LPS and IL-1 beta alone to induce the expression of iNOS, both L PS and IL-1 beta individually stimulated MAP kinase activity and induced DN A binding and transcriptional activity of NF-kappa B, Wortmannin alone was unable to activate MAP kinase and NF-kappa B. Moreover, wortmannin had no e ffect on LPS- or IL-1 beta-mediated activation of MAP kinase and NF-kappa B , suggesting that wortmannin induced the expression of iNOS in LPS- or IL-1 beta-stimulated C-6 glial cells without modulating the activation of MAP k inase and NF-kappa B, Similar to C-6 glial cells, wortmannin also stimulate d LPS-mediated expression of iNOS and production of NO in astrocytes withou t affecting the LPS-mediated activation of NF-kappa B, Taken together, the results from specific chemical inhibitors and dominant-negative mutant expr ession studies demonstrate that apart from the activation of NF-kappa B, in hibition of PI 3-kinase is also necessary for the expression of iNOS and pr oduction of NO.