FIGURE 4.

Interference of NO inhibition with neurosphere differentiation. A and B, gene expression levels of specific markers for mature neurons (MAP-2), glia (GFAP), differentiating neurons (β3-tubulin), and progenitor cells (nestin) of l-NAME-treated (A) and MDLA-treated (B) neurospheres on day 7 of differentiation were determined by real time PCR. Neurospheres were maintained in culture until day 7 of differentiation in the absence or presence of 1 mm l-NAME, a nonselective antagonist of NOS, or 1 mm MDLA, an inhibitor of AS, which were newly supplied every day. The medium was changed every 2 days. Cells differentiated in the absence of these compounds were used as control. C, flow cytometry analysis of Nestin, β3-tubulin, and GFAP expression in neurospheres differentiated for 7 days in the absence or presence of 1 mm MDLA or 1 mm l-NAME. Representative histograms compare expression levels of neural markers in differentiated neurospheres (gray) with neurospheres treated with MDLA (red) or l-NAME (blue). D and E, immunoblots of protein extracts from NSC on day 7 of differentiation cultures in the absence or presence of 1 mm l-NAME (D) or 1 mm MDLA (E) were probed for GFAP and β3-tubulin expression levels. F, flow cytometry analysis of Nestin, β3-tubulin, and GFAP expression in murine neurospheres differentiated for 7 days in the presence of 1 μm 7-Ni. Representative histograms compare expression levels of neural markers in untreated neurospheres (gray) and treated with 7-Ni (green). G, regulation of AS, eNOS and nNOS expression in differentiated neurospheres (day 7) in the presence of activators and inhibitors of the NO-citrulline cycle. H, Western blot analysis was performed to confirm increased eNOS expression in l-NAME-treated neurospheres. The data shown are representative for at least two independent experiments. The data are presented as the mean values ± S.E. ***, p < 0.001. β-tub, β-tubulin, l-cit, l-citrulline.