Inhibition of phosphatidylinositol 3-kinase induces nitric-oxide synthase in lipopolysaccharide- or cytokine-stimulated C6 glial cells

Nitric oxide (NO) produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of demyelinating and neurodegenerative diseases, The present study underlines the importance of phosphatidylinositol 3-kinase (PI 3-kinase) in the expression of iNOS in C-6 glial cells and rat primary astrocytes. Bacterial lipopolysaccharide (LPS) or interleukin-1 beta (IL-1 beta) was unable to induce the expression of iNOS and the production of NO in rat C-6 glial cells. Similarly, wortmannin and LY294002, compounds that inhibit PI3-kinase, were also unable to induce the expression of NOS and the production of NO. However, a combination of wortmannin 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 iNOS, wortmannin also induced iNOS promoter-derived chloramphenicol acetyltransferase activity in LPS- or IL-1 beta-treated C-6 glial cells. The expression of iNOS by LPS in C-6 glial cells expressing a dominant-negative mutant 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 activation of mitogen-activated protein (MAP) kinase and nuclear factor NF-kappa B in LPS- or IL-1 beta-stimulated C-6 glial cells. In contrast to the inability of LPS and IL-1 beta alone to induce the expression of iNOS, both LPS and IL-1 beta individually stimulated MAP kinase activity and induced DNA binding and transcriptional activity of NF-kappa B, Wortmannin alone was unable to activate MAP kinase and NF-kappa B. Moreover, wortmannin had no effect 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 kinase and NF-kappa B, Similar to C-6 glial cells, wortmannin also stimulated LPS-mediated expression of iNOS and production of NO in astrocytes without affecting the LPS-mediated activation of NF-kappa B, Taken together, the results from specific chemical inhibitors and dominant-negative mutant expression studies demonstrate that apart from the activation of NF-kappa B, inhibition of PI 3-kinase is also necessary for the expression of iNOS and production of NO.