The influence of a nucleoside analog 5-aza-2'-deoxycytidine (5-AzadC) on inducible nitric oxide synthase (iNOS)-dependent nitric oxide (NO) production in various rat cell types was investigated. In C6 astrocytoma cell line and primary astrocytes, 5-AzadC enhanced proinflammatory cytokine (IFN-gamma, TNF-alpha, IL-1)-triggered NO synthesis in a time- and dose-dependent manner. In contrast, 5-AzadC did not potentiate NO production in IFN-gamma-stimulated macrophages, fibroblasts, or endothelial cells. Blockade of transcription or translation in C6 cells abolished the observed effect, suggesting the iNOS gene expression, rather than its catalytic activity, as a target for the drug action. Accordingly, 5-AzadC upregulated IFN-gamma-induced expression of iNOS mRNA in C6 astrocytes. The effect of 5-AzadC on astrocyte NO release was blocked by the inhibitor of p44/42 mitogen activated protein kinase-dependent signaling. Finally, the observed stimulatory effect of 5-AzadC on iNOS expression was apparently independent of DNA demethylation, as DNA digestion with methylation-sensitive restriction enzyme HpaII showed that 5-AzadC failed to demethylate cellular DNA in conditions used for iNOS induction.
